F , Mitochondrial potential ( JC - 1 ) . red ( JC - 1 Aggregate ) indicates disrupted mitochondrial potential , green ( JC - 1 Monomer , insets ) indicates intact potential . inset were taken from same microscopic fields of respective JC - 1 aggregate . Mitochondrial damage was induced by treatment with mitochondrial toxins CCCP ( 2µM ) + H2O2 ( 500 µM ) for 4hr . MFIs were estimated in 3 independent cultures ( > 5 randomly selected microscopic fields / culture ) . Data are represented as mean ± . One - way ANOVA . Significantly different at p = 0 . 0006 * , 0 . 0093 # in graph F .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

F, Mitochondrial membrane potential (JC-1). The red fluorescence (JC-1 Aggregate) indicates disrupted mitochondrial membrane potential, and green fluorescence (JC-1 Monomer, insets) indicates intact potential. The inset images were taken from the same microscopic fields of the respective JC-1 aggregate images. Mitochondrial damage was induced by treatment with the mitochondrial toxins CCCP (2µM)+H2O2(500 µM) for 4hr. MFIs were estimated in 3 independent cultures (>5 randomly selected microscopic fields/culture). Data are represented as mean ± SEM. One-way ANOVA. Significantly different at p=0.0006*, 0.0093# in graph F.


G , Intracellular of ATP , an indicator of mitochondrial oxidative phosphorylation . Data are expressed as mean ± . n = 3 cultures ; one - way ANOVA . Significantly different at p = 0 . 0009 * , 0 . 00004 * * , 0 . 04913 # , 0 . 0024 + in graph G .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

G, Intracellular levels of ATP, an indicator of mitochondrial oxidative phosphorylation. Data are expressed as mean ± SEM. n=3 cultures; one-way ANOVA. Significantly different at p= 0.0009*, 0.00004**, 0.04913#, 0.0024+in graph G.


Glial senescence rescued by SGK1 inhibition . ( H ) Expression of genes associated with senescence in - seq data ( VM - glia with vs . without GSK - 650394 treatment ) .

O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Glial cell senescence rescued by SGK1 inhibition. (H) Expression of genes associated with cell senescence in the RNA-seq data (VM-glia with vs. without GSK-650394 treatment).


( I ) Intracellular ROS estimated by DCF - DA . Data are represented as mean ± . n = 3 ; one - way ANOVA . Significantly different at p = 0 . 0074 * , 0 . 012 # in graph I .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(I) Intracellular ROS levels estimated by DCF-DA. Data are represented as mean ± SEM. n=3; one-way ANOVA. Significantly different at p=0.0074*, 0.012# in graph I.


( J ) Senescence - associated β - galactosidase ( SA - β - gal ) activity . Data are represented as mean ± . n = 3 ; one - way ANOVA . Significantly different at p = 2 . 05E - 06 * , 0 . 0042 # , 0 . 0006 + in graph J .

O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(J) Senescence-associated β-galactosidase (SA-β-gal) activity. Data are represented as mean ± SEM. n=3; one-way ANOVA. Significantly different at p=2.05E-06*, 0.0042#, 0.0006+ in graph J.


( K ) Immunoblots for associated with senescence . Significantly different at p = 0 . 008 * , 0 . 001 # ( TNFA ) , 0 . 01 * , 0 . 009 # ( HMGB1 ) , 7 . 32E - 10 * , 1 . 23E - 8 # ( IL6 ) , 9 . 62E - 11 * , 1 . 01E - 5 # ( MMP3 ) , 0 . 001 * , 5 . 72E - 4 # ( LAMIN B ) in graph K .

O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O

(K) Immunoblots for proteins associated with cell senescence. Significantly different at p=0.008*, 0.001# (TNFA), 0.01*, 0.009# (HMGB1), 7.32E-10*, 1.23E-8# (IL6), 9.62E-11*, 1.01E-5# (MMP3), 0.001*, 5.72E-4# (LAMIN B) in graph K.


( L ) IL - 6 secreted from cultured VM - glia . cytokine were estimated using ELISA of media from VM - glia with without SGK1 inhibitor . Data are represented as mean ± . n = 3 cultures . One - way ANOVA . Significantly different at p = 0 . 0028 * , 0 . 0062 # in graph L .

O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(L) IL-6 levels secreted from cultured VM-glia. The cytokine levels were estimated using ELISA of the media from the VM-glia with and without the SGK1 inhibitor. Data are represented as mean ± SEM. n=3 cultures. One-way ANOVA. Significantly different at p=0.0028*, 0.0062# in graph L.


( M ) Glial viability following treatment with senolytic compounds ( azithromycin , fisetin ) . Data are represented as mean ± . n = 3 ; Student ' s t - . Significantly different at p = 0 . 0073 * , 0 . 0018 * * , 0 . 0286 # , 0 . 0269 # # , 0 . 0045 # # # in graph M .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(M) Glial cell viability following treatment with senolytic compounds (azithromycin, fisetin). Data are represented as mean ± SEM. n=3; Student's t-test. Significantly different at p=0.0073*,0.0018**, 0.0286#, 0.0269##, 0.0045### in graph M.


B , Western blot for detecting SNCA oligomers . Data are represented as mean ± . n = 4 ; one - way ANOVA . Significantly different at p = 2 . 5E - 07 * , 1 . 65E - 07 * * , 0 . 022 # , 0 . 029 # # , 0 . 046 + , 0 . 015 + + , 0 . 0136 & , 0 . 0016 & & in graph B .

O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

B, Western blot analysis for detecting SNCA oligomers. Data are represented as mean ± SEM. n=4; one-way ANOVA. Significantly different at p=2.5E-07*, 1.65E-07**, 0.022#, 0.029##, 0.046+, 0.015++, 0.0136&, 0.0016&& in graph B.


C , Immunocytochemical detection of p - 129 - SNCA , associated with SNCA aggregation . Data are represented as mean ± . n = 6 . Significantly different at p = 0 . 0363 * in graph C .

O O O O O O O O O B-MEASURED_VAR O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O

C, Immunocytochemical detection of p-129-SNCA, associated with SNCA aggregation. Data are represented as mean ± SEM. n=6. Significantly different at p=0.0363* in graph C.


E , Bi - FC - assessment of inter - neuronal propagation aggregation of SNCA . Bi - FC + inclusions ( ) were counted in E . Data are represented as mean ± . n = 5 . Significantly different at p = 0 . 0446 * in graph E .

O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

E, Bi-FC-based assessment of the inter-neuronal propagation and aggregation of SNCA. Bi-FC+ inclusions (puncta) were counted in E. Data are represented as mean ± SEM. n=5. Significantly different at p=0.0446* in graph E.


VM - glial cultures were transduced with sh - Sgk1 ( or sh - cont ) lentivirus . Medium was condition in sh - Sgk1 - glia ( or sh - cont - glia ) , conditioned medium ( CM ) treatment effects on neuronal SNCA aggregation were assessed by WB

O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O

VM-glial cultures were transduced with sh-Sgk1 (or sh-cont) lentivirus. Medium was condition in sh-Sgk1-glia (or sh-cont-glia), and the conditioned medium (CM) treatment effects on neuronal SNCA aggregation were assessed by WB


VM - glial cultures were transduced with sh - Sgk1 ( or sh - cont ) lentivirus . Medium was condition in sh - Sgk1 - glia ( or sh - cont - glia ) , conditioned medium ( CM ) treatment effects on neuronal SNCA aggregation were assessed by

O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O


VM - glial cultures were transduced with sh - Sgk1 ( or sh - cont ) lentivirus . Medium was condition in sh - Sgk1 - glia ( or sh - cont - glia ) , conditioned medium ( CM ) treatment effects on neuronal SNCA aggregation p - 129 - SNCA immunocytochemical analyses . Data are represented as mean ± . n = 3 . Significantly different at p = 0 . 00003 * , 0 . 0003 * * , 0 . 0012 * * * , 0 . 00004 * * * * , 0 . 0321 # in graph G p = 0 . 0052 * in graph H .

O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

VM-glial cultures were transduced with sh-Sgk1 (or sh-cont) lentivirus. Medium was condition in sh-Sgk1-glia (or sh-cont-glia), and the conditioned medium (CM) treatment effects on neuronal SNCA aggregation and p-129-SNCA immunocytochemical analyses. Data are represented as mean ± SEM. n=3. Significantly different at p=0.00003*, 0.0003**, 0.0012***, 0.00004****, 0.0321# in graph G and p=0.0052* in graph H.


Glia derived from mouse VM were transduced with sh - Sgk1 ( B ) or SGK1 ( CMV - Sgk1 , C ) , co - cultured with VM - derived mDA neurons . Seven days after co - culture , were exposed to H2O2 ( 500µM , 3hr ) or not ( - ) , then mDA neuron viability ( TH + ) neurite degeneration ( TH + fiber lengths ) were estimated . Data are represented as mean ± ; n = 4 culture coverslips . Student ' s t - . Significantly different at p = 0 . 033 * , 0 . 0412 * * , 7 . 44E - 23 # , 1 . 39E - 23 # # in graph B p = 0 . 0489 * , 0 . 0366 * * , 0 . 0078 # # in graph C .

O O O O O O O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Glia derived from mouse VM were transduced with sh-Sgk1 (B) or SGK1 (CMV-Sgk1, C), and co-cultured with VM-derived mDA neurons. Seven days after co-culture, cells were exposed to H2O2 (500µM, 3hr) or not (-), and then mDA neuron viability (TH+ cell counts) and neurite degeneration (TH+ fiber lengths) were estimated. Data are represented as mean ± SEM; n=4 culture coverslips. Student's t-test. Significantly different at p=0.033*, 0.0412**, 7.44E-23#,1.39E-23## in graph B and p=0.0489*, 0.0366**, 0.0078## in graph C.


Glial neurotrophic functions potentiated by SGK1 downregulation were mediated in a paracrine manner . VM - glia transduced with sh - Sgk1 ( or sh - cont as a control ) were challenged with H2O2 + LPS for 3hr or not . Glial conditioned medium ( GCM ) was prepared in each glial culture condition administered to mDA neurons primarily cultured from mouse VM ( E ) viability of neuronal was estimated using CCK8 assays TuJ1 + . Data are represented as mean ± . n = 3 cultures ; One - way ANOVA . Significantly different at p = 0 . 0002 * , 3 . 29E - 08 * * , 0 . 0003 * * * , 3 . 66E - 06 # , 0 . 0029 # # ( TuJ1 + counting ) , 0 . 0127 + , 5 . 98E - 06 + + , 0 . 0002 + + + , 0 . 0005 & , 0 . 0078 & & ( CCK8 assays ) in graph E .

O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Glial neurotrophic functions potentiated by SGK1 downregulation were mediated in a paracrine manner. VM-glia transduced with sh-Sgk1 (or sh-cont as a control) were challenged with H2O2+LPS for 3hr or not. Glial conditioned medium (GCM) was prepared in each glial culture condition and administered to mDA neurons primarily cultured from mouse VM (E) Cell viability of the neuronal cells was estimated using CCK8 assays and TuJ1+ cell counts. Data are represented as mean ± SEM. n=3 cultures; One-way ANOVA. Significantly different at p=0.0002*, 3.29E-08**, 0.0003***, 3.66E-06#, 0.0029## (TuJ1+ cell counting), 0.0127+, 5.98E-06++, 0.0002+++, 0.0005&, 0.0078&& (CCK8 assays) in graph E.


F , Effect of SGK1 inhibitor treatment in mixed mDA neuron + VM - glia co - cultures . Data are represented as mean ± . n = 3 . Student ' s t - . Significantly different at p = 0 . 0068 * , 0 . 0368 * * , 0 . 0249 # , 1 . 48E - 27 # # in graph F .

O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

F, Effect of the SGK1 inhibitor treatment in the mixed mDA neuron + VM-glia co-cultures. Data are represented as mean ± SEM. n=3. Student's t-test. Significantly different at p=0.0068*, 0.0368**, 0.0249#, 1.48E-27## in graph F.


( B - D ) Behaviors of MPTP - PD mice assessed by pole ( B ) , beam ( C ) at 3 - 6 post - MPTP injection , locomotor activity ( total & average speed , D ) at 6 post - MPTP injection . n = 20 GSK - 650394 - treated MPTP - PD mice , 14 vehicle - treated MPTP - PD mice , 8 MPTP - untreated non - PD mice . Data are represented as mean ± . Significantly different at p = 0 . 0012 # , 0 . 0001 * , 3 . 09E - 08 # # , 8 . 21E - 08 * * in graph B p = 0 . 0009 # , 0 . 0035 * , 1 . 03E - 06 # # , 0 . 00003 * * in graph C p = 0 . 0001 * , 0 . 0284 * * , 0 . 0101 # ( total ) , 1 . 49E - 08 * , 0 . 0005 * * , 0 . 0001 # ( average speed ) in graph D .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(B-D) Behaviors of MPTP-PD mice assessed by pole (B), beam (C) at 3-6 weeks post-MPTP injection, and locomotor activity (total distance & average speed, D) tests at 6 weeks post-MPTP injection. n= 20 GSK-650394-treated MPTP-PD mice, 14 vehicle-treated MPTP-PD mice, and 8 MPTP-untreated non-PD mice. Data are represented as mean ± SEM. Significantly different at p=0.0012#, 0.0001*, 3.09E-08##, 8.21E-08** in graph B and p=0.0009#, 0.0035*, 1.03E-06##, 0.00003** in graph C and p=0.0001*, 0.0284**, 0.0101# (total distance), 1.49E-08*, 0.0005**, 0.0001# (average speed) in graph D.


Histologic assessment of number of TH + mDA neurons ( E , F ) , neurite lengths ( E , G ) , sizes ( E , H ) Shown are representative for TH + DA neurons in SN TH + DA neurons in boxed areas of ( E ) are enlarged in right .

O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O

Histologic assessment of the number of TH+ mDA neurons (E, F), neurite lengths (E, G), and cell body sizes (E, H) Shown are the representative images for TH+ DA neurons in the SN TH+ DA neurons in boxed areas of (E) are enlarged in right.


Histologic assessment of number of TH + mDA neurons in SN TH + fiber intensities in striatum Shown are representative for TH + DA neurons in TH - immunoreactivity in striatum ,

O O O O O B-MEASURED_VAR O O O O O B-MEASURED_VAR O O O O O O O O O B-MEASURED_VAR O O O O B-MEASURED_VAR O O O O O

Histologic assessment of the number of TH+ mDA neurons in the SN and TH+ fiber intensities in the striatum Shown are the representative images for TH+ DA neurons in the TH-immunoreactivity in the striatum,


Therapeutic effects of GSK - 650394 in SNCA - PD model mice . Behaviors ( L - N ) of SNCA - PD mice were assessed at 6 - 12 post - SNCA injection Significantly different at p = 0 . 008 # , 0 . 021 * , 0 . 00001 # # , 0 . 0009 * * in graph L p = 0 . 00002 # , 0 . 0344 * , 0 . 0005 # # , 0 . 0067 * * in graph M p = 0 . 0057 # , 0 . 0345 * ( total ) , 0 . 0001 # , 0 . 0375 # # , 0 . 0338 * ( average speed ) in graph N .

O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Therapeutic effects of GSK-650394 in SNCA-PD model mice. Behaviors (L-N) of the SNCA-PD mice were assessed at 6-12 weeks post- SNCA injection Significantly different at p=0.008#, 0.021*, 0.00001##, 0.0009** in graph L and p=0.00002#, 0.0344*, 0.0005##, 0.0067** in graph M and p=0.0057#, 0.0345* (total distance), 0.0001#, 0.0375##, 0.0338* (average speed) in graph N.


Therapeutic effects of GSK - 650394 in SNCA - PD model mice . mDA neuron degeneration in SN of SNCA - PD mice were assessed at 12 post - SNCA injection after sacrifice SNCA pathology assessed by p129 - α - syn immunoreactivity in TH + mDA neurons in SN . n = 5 GSK - 650394 - treated SNCA - PD mice , 5 vehicle - treated SNCA - PD mice , 8 SNCA - untreated non - PD mice .

O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O B-MEASURED_VAR O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O O O O O O O

Therapeutic effects of GSK-650394 in SNCA-PD model mice. mDA neuron degeneration in the SN of the SNCA-PD mice were assessed at 12 weeks post- SNCA injection after sacrifice SNCA pathology assessed by p129-α-syn immunoreactivity in TH+ mDA neurons in the SN. n= 5 GSK-650394-treated SNCA-PD mice, 5 vehicle-treated SNCA-PD mice, and 8 SNCA-untreated non-PD mice.


Therapeutic effects of GSK - 650394 in SNCA - PD model mice . mDA neuron degeneration in SN ( O - U ) of SNCA - PD mice were assessed 12 post - SNCA injection after sacrifice SNCA pathology assessed by p129 - α - syn immunoreactivity in TH + mDA neurons in SN . n = 5 GSK - 650394 - treated SNCA - PD mice , 5 vehicle - treated SNCA - PD mice , 8 SNCA - untreated non - PD mice .

O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O B-MEASURED_VAR O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O O O O O O O

Therapeutic effects of GSK-650394 in SNCA-PD model mice. mDA neuron degeneration in the SN (O-U) of the SNCA-PD mice were assessed 12 weeks post- SNCA injection after sacrifice SNCA pathology assessed by p129-α-syn immunoreactivity in TH+ mDA neurons in the SN. n= 5 GSK-650394-treated SNCA-PD mice, 5 vehicle-treated SNCA-PD mice, and 8 SNCA-untreated non-PD mice.


A B , Efficiency of AAV9 - mediated gene delivery . AA9 expressing reporter GFP gene were stereotaxically injected into SN of mice , GFP expression in astrocytes , microglia , neuronal was quantified . Data are represented as mean ± . n = 3 mice ( > 5 randomly selected microscopic fields per were counted ) . Significantly different at p = 0 . 006 * , 5 . 1E - 09 # , 5 . 36E - 09 + in graph B .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A and B, Efficiency of AAV9-mediated gene delivery. AA9 viruses expressing the reporter GFP gene were stereotaxically injected into the SN of mice, and GFP expression in astrocytes, microglia, and neuronal cells was quantified. Data are represented as mean ± SEM. n=3 mice (>5 randomly selected microscopic fields per animal were counted). Significantly different at p=0.006*, 5.1E-09#, 5.36E-09+ in graph B.


SGK1 knockdown effect in vivo by treatment with shSgk1 - AAV9 . Total SGK1 mRNA were estimated in midbrain SN of PD mice injected with shSgk1 ( shControl as control ) AAV9 using real time - PCR ( D ) .

B-MEASURED_VAR O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O

SGK1 knockdown effect in vivo by treatment with shSgk1-AAV9. Total SGK1 mRNA levels were estimated in the midbrain SN of the PD mice injected with shSgk1(shControl as control) AAV9 using real time-PCR (D).


SGK1 knockdown effect in vivo by treatment with shSgk1 - AAV9 . Total

SGK1 knockdown effect in vivo by treatment with shSgk1-AAV9. Total SGK1


type - specific SGK1 knockdown was estimated by counting SGK1 - immuonoreactivity in neuron ( MAP2 + ) , astrocyte ( GFAP + ) , microglia ( Iba1 + ) populations ( E ) .

O O O B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O O O O O B-MEASURED_VAR O O O O O B-MEASURED_VAR O O O O O B-MEASURED_VAR O O O O O O O

Cell type-specific SGK1 knockdown was estimated by counting SGK1-immuonoreactivity in neuron (MAP2+), astrocyte (GFAP+), and microglia (Iba1+) populations (E).


Data are represented as mean ± . n = 4 mice in each group . Significantly different at p = 0 . 0004 # in graph D p = 0 . 049 * , 0 . 007 * * , 0 . 002 * * * in graph E . ( F - H ) Behavior of PD mice assessed by pole ( F ) , beam ( G ) at 6 - 12 , locomotor activity ( H ) at 12 post - SNCA injection . Significantly different at p = 0 . 0003 # , 0 . 0428 * , 7 . 46E - 06 # # , 0 . 0012 * * in graph F p = 8 . 04E - 08 # , 0 . 004 * , 1 . 62E - 09 # # , 3 . 3E - 06 * * in graph G p = 0 . 0002 # , 0 . 0194 * ( total ) , 9 . 54E - 06 # , 0 . 0047 * ( average speed ) in graph H .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Data are represented as mean ± SEM. n=4 mice in each group. Significantly different at p=0.0004# in graph D and p=0.049*, 0.007**, 0.002*** in graph E. (F-H) Behavior of the PD mice assessed by pole (F), beam (G) at 6-12 weeks, and locomotor activity (H) tests at 12 weeks post- SNCA injection. Significantly different at p=0.0003#, 0.0428*, 7.46E-06##, 0.0012** in graph F and p=8.04E-08#, 0.004*, 1.62E-09##, 3.3E-06** in graph G and p=0.0002#, 0.0194* (total distance), 9.54E-06#, 0.0047* (average speed) in graph H.


Histologic assessment of p129 - SNCA immunoreactivity ( I , J ) , TH + mDA neuron ( I , K ) , neurite lengths ( L ) , sizes ( M ) in SN Significantly different at p = 0 . 0001 * in graph J , p = 2 . 35E - 25 # , 1 . 09E - 16 # # , 4 . 36E - 08 * in graph K p = 1 . 5E - 40 # , 1 . 08E - 24 # # , 2 . 32E - 07 * in graph L p = 9 . 07E - 20 # , 2 . 59E - 16 # # , 0 . 05 * in graph M p = 4 . 62E - 22 # , 5 . 11E - 12 # # , 3 . 49E - 06 * ( TH ) , n = 7 sh - SGK1 - AAV9 - treated PD mice , 7 sh - cont - AAV9 - treated PD mice , 8 SNCA - untreated mice . Data are represented as mean ± . One - way ANOVA . Scale bar , 100µm .

O O O O O B-MEASURED_VAR O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O

Histologic assessment of p129-SNCA immunoreactivity ( I,J), TH+ mDA neuron counts (I,K), neurite lengths (L), and cell body sizes (M) in the SN Significantly different at p=0.0001* in graph J, p=2.35E-25#, 1.09E-16##, 4.36E-08* in graph K and p=1.5E-40#, 1.08E-24##, 2.32E-07* in graph L and p=9.07E-20#, 2.59E-16##, 0.05* in graph M and p=4.62E-22#, 5.11E-12##, 3.49E-06* (TH), n= 7 sh-SGK1-AAV9-treated PD mice, 7 sh-cont-AAV9-treated PD mice, and 8 SNCA-untreated mice. Data are represented as mean ± SEM. One-way ANOVA. Scale bar, 100µm.


Histologic assessment of TH + DA transporter ( DAT ) fiber intensities in striatum n = 7 sh - SGK1 - AAV9 - treated PD mice , 7 sh - cont - AAV9 - treated PD mice , 8 SNCA - untreated mice . Data are represented as mean ± . One - way ANOVA . Scale bar , 100µm .

O O O B-MEASURED_VAR O B-MEASURED_VAR I-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O

Histologic assessment of TH+ and DA transporter (DAT) fiber intensities in the striatum n= 7 sh-SGK1-AAV9-treated PD mice, 7 sh-cont-AAV9-treated PD mice, and 8 SNCA-untreated mice. Data are represented as mean ± SEM. One-way ANOVA. Scale bar, 100µm.


A - C , Quantitative morphometric analyses of microglia in SN of SNCA - PD mice . Shown in A are representative views of Iba1 + microglia neighboring TH + mDA neurons in SNs of PD mice treated with GSK - 650394 vehicle . Insets , enlarged views for boxed areas . Data are represented as mean ± . n = 3 mice per group . Significantly different at p = 1 . 49E - 11 * in graph B p = 0 . 000003 * in graph C .

O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A-C, Quantitative and morphometric analyses of microglia in the SN area of SNCA-PD mice. Shown in A are representative views of Iba1+ microglia neighboring TH+ mDA neurons in the SNs of PD mice treated with GSK-650394 and vehicle. Insets, enlarged views for the boxed areas. Data are represented as mean ± SEM. n=3 mice per group. Significantly different at p=1.49E-11* in graph B and p=0.000003* in graph C.


D E , M1 marker CD16 / 32 expression in Iba1 + microglia . Data are represented as mean ± . n = 3 mice per group . Significantly different at p = 0 . 0016 * in graph E .

O O O O O B-MEASURED_VAR O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

D and E, The M1 marker CD16/32 expression in Iba1+ microglia. Data are represented as mean ± SEM. n=3 mice per group. Significantly different at p=0.0016* in graph E.


F G , mRNA expression of NLRP3 - inflammasome components pro - inflammatory cytokines in MPTP ( F ) SNCA ( G ) PD mice injected with SGK1 inhibitor or vehicle ( control ) . Each bar represents mean ± of 3 PCR values from each , n = 3 mice per group . Significantly different at p = 0 . 0372 * , 0 . 0173 * * , 0 . 0003 * * * in graph F p = 0 . 002 # , 0 . 0113 # # , 0 . 0053 # # # , 0 . 0001 # # # # in graph G .

O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

F and G, mRNA expression of NLRP3-inflammasome components and pro-inflammatory cytokines in MPTP (F) and SNCA (G) PD mice injected with the SGK1 inhibitor or vehicle (control). Each bar represents the mean ± SEM of 3 PCR values from each animal, n=3 mice per group. Significantly different at p=0.0372*, 0.0173**, 0.0003*** in graph F and p=0.002#, 0.0113##, 0.0053###, 0.0001#### in graph G.


H - J , ASC expression in SNs SN microglia of SNCA - PD mice . n = 5 mice per group . Data are represented as mean ± . Significantly different at p = 1 . 91E - 09 * in graph I p = 0 . 0079 * in graph J .

O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

H-J, ASC expression in the SNs and SN microglia of SNCA-PD mice. n=5 mice per group. Data are represented as mean ± SEM. Significantly different at p=1.91E-09* in graph I and p=0.0079* in graph J.


K L , β - gal - stained senescence in SNCA - PD mouse SN . Data are represented as mean ± . n = 5 mice per group . Significantly different at p = 0 . 0001 *

O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

K and L, β-gal-stained cell senescence in SNCA-PD mouse SN. Data are represented as mean ± SEM. n=5 mice per group. Significantly different at p=0.0001*


( a ) Mitochondrial tubules in HeLa ( top , TMRE stained ) exceeded dimensions of that for autophagosomes ( labelled by EGFP - LC3B ) under either normal ( middle , same field as TMRE image ) or starvation conditions ( right ) .

O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O

(a) Mitochondrial tubules in HeLa cells (top, TMRE stained) exceeded the dimensions of that for autophagosomes (labelled by EGFP-LC3B) under either normal (middle, same field as TMRE image) or starvation conditions (right).


( c ) Illuminating a selective region ( dotted circle , top ) within a KR - dMito / EBFP2 - parkin - expressing HeLa led to immediate loss of KR ( top two panels ) . Third to fifth row : magnified view of photo - impaired region , EBFP2 - parkin translocated onto impaired mitochondrial tubules synchronously ( 38 - 60 min after 559 illumination , arrows ) . Scale bars ( panels ) , 10 μm .

O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(c) Illuminating a selective region (white dotted circle, top) within a KR-dMito/EBFP2-parkin-expressing HeLa cell led to the immediate loss of KR fluorescence (top two panels). Third to fifth row: magnified view of the photo-impaired region, EBFP2-parkin translocated onto impaired mitochondrial tubules synchronously (38-60 min after 559 illumination, white arrows). Scale bars (all panels), 10 μm.


( a ) A HeLa expressing KR - dMito , EGFP - LC3B EBFP2 - parkin ( cyan ) were 559 nm illuminated ( dotted circle region ; immediate loss of KR ) . ( b - d ) Magnified view of photo - inactivated region in a 34 - 93 min after 559 nm illumination . ( b ) Induction of parkin - mediated mitophagy on a single , long mitochondria tubule ( arrows ) . ( c ) LC3B - coated structures were recruited onto distinct spots on photo - impaired mitochondrial tubule ( arrows : location of original impaired single mitochondrial tubule ) . ( d ) dotted region outlined in c , LC3 structures segregated from main parkin - labelled cluster , carrying away bits of parkin - labelled mitochondria with them . Two events are labelled in image with yellow arrows , respectively . Scale bars ( panels ) , 5 μm .

O O O O O O O O B-MEASURED_VAR O O O B-MEASURED_VAR O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O B-MEASURED_VAR O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(a) A HeLa cell expressing KR-dMito, EGFP-LC3B and EBFP2-parkin (cyan) were 559 nm illuminated (white dotted circle region; immediate loss of KR fluorescence). (b-d) Magnified view of the photo-inactivated region in a 34-93 min after 559 nm illumination. (b) Induction of parkin-mediated mitophagy on a single, long mitochondria tubule (white arrows). (c) LC3B-coated structures were recruited onto distinct spots on the photo-impaired mitochondrial tubule (white arrows: the location of the original impaired single mitochondrial tubule). (d) White dotted line region outlined in c, LC3 structures segregated from the main parkin-labelled cluster, carrying away bits of the parkin-labelled mitochondria with them. Two individual events are labelled in the image sequence with white and yellow arrows, respectively. Scale bars (all panels), 5 μm.


( a ) A HeLa expressing KR - dMito ( red ) , EGFP - DFCP1 ( green ) EBFP2 - parkin ( cyan ) were illuminated with 559 nm at dotted circle region , leading to generation of a long parkin - labelled mitochondria tubule ( right , magnified view of - activated region ) for autophagic turnover ( arrow ) . Scale bars , 10 μm .

O O O O O O O O B-MEASURED_VAR O O O O O O B-MEASURED_VAR O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(a) A HeLa cell expressing KR-dMito (red), EGFP-DFCP1 (green) and EBFP2-parkin (cyan) were illuminated with 559 nm at the white dotted circle region, leading to the generation of a long parkin-labelled mitochondria tubule (right, magnified view of the light-activated region) for autophagic turnover (white arrow). Scale bars, 10 μm.


( b ) After generation of parkin - labelled mitochondrial tubules following treatments in a , discrete DFCP1 foci ( green , middle panel , ER - associated omegasomes ) formed along parkin - labelled mitochondria ( arrows indicate DFCP1 spots that colocalized with parkin ) . Bottom panel : merged image of parkin DFCP1 . Scale bar , 10 μm .

O O O O O O B-MEASURED_VAR O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O B-MEASURED_VAR O O O O B-MEASURED_VAR O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O

(b) After the generation of parkin-labelled mitochondrial tubules following treatments in a, discrete DFCP1 foci (green, middle panel, ER-associated omegasomes) formed along the parkin-labelled mitochondria (white arrows indicate DFCP1 spots that colocalized with parkin). Bottom panel: merged image of parkin and DFCP1. Scale bar, 10 μm.


( c ) Time - lapse imaging showing DCFP1 formation on parkin - labelled mitochondria . A HeLa expressing KR - dMito , EGFP - DFCP1 ( green ) EBFP2 - parkin ( pseudo - coloured red ) were 559 nm illuminated , leading to generation of parkin - labelled mitochondria tubules . Time - lapse imaging ( 1 frame per min ) revealed that DFCP1 ( green ) formed transiently at discrete sites on parkin - labelled mitochondrial tubules ( red ) . arrows mark two DFCP1 ( ER - associated omegasomes ) formation / disassembly events directly on parkin - labelled tubules . Scale bar , 5 μm .

O O O O O O O O B-MEASURED_VAR O O B-MEASURED_VAR O O O O O O O O O B-MEASURED_VAR O O O B-MEASURED_VAR O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O

(c) Time-lapse imaging showing DCFP1 puncta formation on parkin-labelled mitochondria. A HeLa cell expressing KR-dMito, EGFP-DFCP1 (green) and EBFP2-parkin (pseudo-coloured red) were 559 nm illuminated, leading to the generation of parkin-labelled mitochondria tubules. Time-lapse imaging (1 frame per min) revealed that DFCP1 puncta (green) formed transiently at discrete sites on parkin-labelled mitochondrial tubules (red). White arrows mark two DFCP1 puncta (ER-associated omegasomes) formation/disassembly events directly on parkin-labelled tubules. Scale bar, 5 μm.


( d ) A COS - 7 expressing EBFP2 - parkin ( cyan ) , AcGFP - sec61β ( green ) TagRFP - DFCP1 ( red ) were treated with 10 μM CCCP for 4 h imaged . Omegasomes ( DFCP1 ) appeared where ER ( sec61β ) impaired mitochondria ( parkin ) overlap . Scale bar , 10 μm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O B-MEASURED_VAR O O O O B-MEASURED_VAR O O O O O O O O O O

(d) A COS-7 cell expressing EBFP2-parkin (cyan), AcGFP-sec61β (green) and TagRFP-DFCP1 (red) were treated with 10 μM CCCP for 4 h and imaged. Omegasomes (DFCP1) appeared where ER (sec61β) and impaired mitochondria (parkin) overlap. Scale bar, 10 μm.


( e ) Histogram of lifetime of DFCP1 foci on parkin - labelled mitochondria in CCCP - treated HeLa expressing TagRFP - DFCP1 , EGFP - LC3B EBFP2 - parkin .

O O O O O O O B-MEASURED_VAR O O B-MEASURED_VAR O O O O O O O O O O O B-MEASURED_VAR O O O B-MEASURED_VAR O O B-MEASURED_VAR O

(e) Histogram of the lifetime of individual DFCP1 foci on parkin-labelled mitochondria in CCCP-treated HeLa cells expressing TagRFP-DFCP1, EGFP-LC3B and EBFP2-parkin.


( f ) Wortmannin treatments delayed initial LC3 recruitment times onto parkin - labelled mitochondria in HeLa expressing KR - dMito , EGFP - LC3B EBFP2 - parkin . Each ' + ' marks times measured in HeLa ( red ' □ ' ( square ) denotes average from five ) . P = 0 . 0172 ( Student ' s t - ) .

O O O O O O O B-MEASURED_VAR O O O B-MEASURED_VAR O O O O O O O O B-MEASURED_VAR O O O B-MEASURED_VAR O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(f) Wortmannin treatments delayed the initial LC3 recruitment times onto parkin-labelled mitochondria in HeLa cells expressing KR-dMito, EGFP-LC3B and EBFP2-parkin. Each '+' marks the times measured in individual HeLa cells (the red '□' (square) denotes the average from five cells). P=0.0172 (Student's t-test).


( a ) A COS - 7 expressing EBFP2 - Ub ( red ) , AcGFP - sec61β ( green ) mcherry - parkin ( cyan ) was treated with 10 μM CCCP for 1 h imaged . Impaired mitochondria ( parkin ) displayed preferentially ubiquitinated foci ( arrows ) at selected regions that overlap with ER ( sec61β ) .

O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O

(a) A COS-7 cell expressing EBFP2-Ub (red), AcGFP-sec61β (green) and mcherry-parkin (cyan) was treated with 10 μM CCCP for 1 h and imaged. Impaired mitochondria (parkin) displayed preferentially ubiquitinated foci (white arrows) at selected regions that overlap with the ER (sec61β).


( b ) HeLa expressing KR - dMito ( red ) , EGFP - LC3 ( green ) EBFP2 - parkin ( cyan ) were illuminated with 559 nm at dotted square regions , generating parkin - labelled mitochondria ( bottom right , magnified view of - activated regions ) . ( c ) Magnified view of photo - inactivated region in b 80 min after illumination ( post - stained with anti - ubiquitin antibody ) . LC3 ubiquitin colocalized at discrete sites along parkin - labelled mitochondria ( arrows ) .

O O O O O O O B-MEASURED_VAR O O O O O O B-MEASURED_VAR O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O B-MEASURED_VAR O O O O O O O

(b) HeLa cells expressing KR-dMito (red), EGFP-LC3 (green) and EBFP2-parkin (cyan) were illuminated with 559 nm at the white dotted square regions, generating parkin-labelled mitochondria (bottom right, magnified view of the light-activated regions). (c) Magnified view of the photo-inactivated region in b 80 min after illumination (post-stained with anti-ubiquitin antibody). LC3 and ubiquitin colocalized at discrete sites along parkin-labelled mitochondria (white arrows).


( d ) A MG - 132 treated HeLa expressing KR - dMito , EBFP2 - parkin ( green ) EGFP - LC3B ( red ) was 559 nm illuminated , leading to appearance of parkin - labelled mitochondria ( arrows one to three indicated structures , top panels ) . parkin - labelled structures did not fragment nor became engulfed by LC3 structures ( bottom panels ) in presence MG - 132 . Scale bars ( panels ) , 5 μm .

O O O O O O O O O O O O B-MEASURED_VAR O O O B-MEASURED_VAR O O O O O B-MEASURED_VAR O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O

(d) A MG-132 treated HeLa cell expressing KR-dMito, EBFP2-parkin (green) and EGFP-LC3B (red) was 559 nm illuminated, leading to the appearance of parkin-labelled mitochondria (white arrows one to three indicated structures, top panels). The parkin-labelled structures did not fragment nor became engulfed by LC3 structures (bottom panels) in the presence MG-132. Scale bars (all panels), 5 μm.


A . Confocal of mitochondrial in wild - type ( WT ) Drp1 - / - 293T transfected with empty vector ( left panel ) Myc - hFis1 ( right panel ) , stained with MitoTracker ( red ) followed by immunostaining with anti - Myc antibody ( green ) .

O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O

A. Confocal images of mitochondrial morphology in wild-type (WT) and Drp1-/- 293T cells transfected with empty vector (left panel) and Myc-hFis1 (right panel), stained with MitoTracker (red) followed by immunostaining with anti-Myc antibody (green).


B . Quantitative analyses of fragmented mitochondria ( mean ( μm2 ) per mitochondrion ) after Myc - hFis1 overexpression in WT Drp1 - / - 293T using Image J software ( Particle ) in three independent experiments . In each , only dispersed mitochondria were analyzed . total number of mitochondria ( mito ) analyzed is indicated for each condition .

O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

B. Quantitative analyses of fragmented mitochondria size (mean area (μm2) per mitochondrion) after Myc-hFis1 overexpression in WT and Drp1-/- 293T cells using Image J software (Particle analysis) in three independent experiments. In each cell, only dispersed individual mitochondria were analyzed. The total number of mitochondria (mito) analyzed is indicated for each condition.


C . Quantitative of mean mitochondria number per in WT Drp1 - / - 293T overexpressing Myc - hFis1 using Image J software ( Particle ) in three independent experiments .

O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O

C. Quantitative analysis of mean mitochondria number per cell in WT and Drp1-/- 293T cells overexpressing Myc-hFis1 using Image J software (Particle analysis) in three independent experiments.


E . Percentages of with indicated mitochondrial morphologies in WT Drp1 - / - 293T transfected with empty vector ( control ) , Myc - hFis1 either Myc - or GFP - tagged mutants as indicated in three independent experiments .

O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O

E. Percentages of cells with indicated mitochondrial morphologies in WT and Drp1-/- 293T cells transfected with empty vector (control), Myc-hFis1 and either Myc- or GFP-tagged mutants as indicated in three independent experiments.


A . Confocal of mitochondrial in WT 293T treated with scrambled siRNA ( control ) Dyn2 siRNA as indicated , followed by transfection with empty vector ( left panel ) Myc - hFis1 ( right three panels ) as indicated . were stained with MitoTracker ( red ) followed by immunostaining with anti - Myc antibody ( green ) .

O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O

A. Confocal images of mitochondrial morphology in WT 293T cells treated with scrambled siRNA (control) and Dyn2 siRNA as indicated, followed by transfection with empty vector (left panel) and Myc-hFis1 (right three panels) as indicated. Cells were stained with MitoTracker (red) followed by immunostaining with anti-Myc antibody (green).


B . Western blot of Dyn2 , Myc - hFis1 , Drp1 GAPDH in WT 293T collected from experiments in ( A ) .

O O O O O B-MEASURED_VAR O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O

B. Western blot of Dyn2, Myc-hFis1, Drp1 and GAPDH in WT 293T cells collected from the experiments in (A).


C . Percentages of with indicated mitochondrial morphologies in WT 293T treated with scrambled siRNA Dyn2 siRNA as indicated , followed by transfection with empty vector or Myc - hFis1 in three independent experiments .

O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O

C. Percentages of cells with indicated mitochondrial morphologies in WT 293T cells treated with scrambled siRNA and Dyn2 siRNA as indicated, followed by transfection with empty vector or Myc-hFis1 in three independent experiments.


D . Confocal of mitochondrial in Drp1 - / - 293T treated with scrambled siRNA Dyn2 siRNA as indicated , followed by transfection with empty vector ( left panel ) Myc - hFis1 ( right three panels ) . were stained with MitoTracker ( red ) followed by immunostaining with anti - Myc antibody ( green ) . Representative examples of tubular , fragmented tubular cluster are indicated .

O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

D. Confocal images of mitochondrial morphology in Drp1-/- 293T cells treated with scrambled siRNA and Dyn2 siRNA as indicated, followed by transfection with empty vector (left panel) and Myc-hFis1 (right three panels). Cells were stained with MitoTracker (red) followed by immunostaining with anti-Myc antibody (green). Representative examples of tubular, fragmented and tubular cluster phenotypes are indicated.


E . Western blot of Dyn2 , Myc - hFis1 GAPDH in Drp1 - / - 293T collected from experiments in ( D ) .

O O O O O B-MEASURED_VAR O O O B-MEASURED_VAR O O O O O O O O O O O O O O O

E. Western blot of Dyn2, Myc-hFis1 and GAPDH in Drp1-/- 293T cells collected from the experiments in (D).


F . Percentages of with indicated mitochondrial morphologies in Drp1 - / - 293T treated with scrambled siRNA or Dyn2 siRNA , followed by transfection with empty vector or Myc - hFis1 as indicated in three independent experiments .

O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O

F. Percentages of cells with indicated mitochondrial morphologies in Drp1-/- 293T cells treated with scrambled siRNA or Dyn2 siRNA, followed by transfection with empty vector or Myc-hFis1 as indicated in three independent experiments.


A . Confocal of mitochondrial in Drp1 - / - 293T transfected with scrambled siRNA ( control ) or with hFis1 - siRNA then stained with MitoTracker ( red ) .

O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O

A. Confocal images of mitochondrial morphology in Drp1-/- 293T cells transfected with scrambled siRNA (control) or with hFis1-siRNA and then stained with MitoTracker (red).


B . Confocal of mitochondrial in Drp1 - / - 293T transfected with either Mfn1 - Myc , Mfn2 - Myc or OPA1 - Myc , stained with MitoTracker ( red ) followed by immunostaining with anti - Myc antibody ( green ) .

O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O

B. Confocal images of mitochondrial morphology in Drp1-/- 293T cells transfected with either Mfn1-Myc, Mfn2-Myc or OPA1-Myc, stained with MitoTracker (red) followed by immunostaining with anti-Myc antibody (green).


C . Percentages ( mean ± s . e . m . ) of with indicated mitochondrial morphologies in Drp1 - / - 293T transfected with either scrambled siRNA ( Ctr ) , hFis1 siRNA , Mfn1 - Myc , Mfn2 - Myc or OPA1 - Myc in three independent experiments for each condition ( n represents number of analyzed ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O

C. Percentages (mean ± s.e.m.) of cells with indicated mitochondrial morphologies in Drp1-/- 293T cells transfected with either scrambled siRNA (Ctr), hFis1 siRNA, Mfn1-Myc, Mfn2-Myc or OPA1-Myc in three independent experiments for each condition (n represents the number of cells analyzed).


D . hFis1 interacts with Mfn1 , Mfn2 OPA1 as well as Drp1 , but not with Dyn2 at endogenous following chemical crosslinking . Wild - type ( WT ) Drp1 - / - 293T were in vivo crosslinked with 1 % formaldehyde ( FA ) lysates were used for co - immunoprecipitation ( IP ) with G beads bound to rabbit normal IgG ( negative control ) or rabbit anti - hFis1 antibody as indicated , followed by immunoblotting with indicated antibodies .

O O B-MEASURED_VAR O O B-MEASURED_VAR O B-MEASURED_VAR I-MEASURED_VAR O O O B-MEASURED_VAR O O O O B-MEASURED_VAR O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O

D. hFis1 interacts with Mfn1, Mfn2 and OPA1 as well as Drp1, but not with Dyn2 at endogenous levels following chemical crosslinking. Wild-type (WT) and Drp1-/- 293T cells were in vivo crosslinked with 1% formaldehyde (FA) and cell lysates were used for co-immunoprecipitation (IP) with Protein G beads bound to rabbit normal IgG (negative control) or rabbit anti-hFis1 antibody as indicated, followed by immunoblotting with indicated antibodies.


hFis1 binds to Mfn1 , Mfn2 OPA1 at endogenous also in absence of chemical crosslinking . lysates prepared from WT 293T ( E ) without chemical crosslinking were used for co - immunoprecipitation ( IP ) with G beads bound to rabbit normal IgG ( negative control ) or rabbit anti - hFis1 antibody as indicated , followed by Western blotting with indicated antibodies .

B-MEASURED_VAR O O B-MEASURED_VAR O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O

hFis1 binds to Mfn1, Mfn2 and OPA1 at endogenous levels also in the absence of chemical crosslinking. Cell lysates prepared from WT 293T (E) cells without chemical crosslinking were used for co-immunoprecipitation (IP) with Protein G beads bound to rabbit normal IgG (negative control) or rabbit anti-hFis1 antibody as indicated, followed by Western blotting with indicated antibodies.


F . hFis1 binds to Mfn1 , Mfn2 OPA1 at endogenous also in absence of chemical crosslinking . lysates prepared from HeLa ( F ) without chemical crosslinking were used for co - immunoprecipitation ( IP ) with G beads bound to rabbit normal IgG ( negative control ) or rabbit anti - hFis1 antibody as indicated , followed by Western blotting with indicated antibodies .

O O B-MEASURED_VAR O O B-MEASURED_VAR O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O

F. hFis1 binds to Mfn1, Mfn2 and OPA1 at endogenous levels also in the absence of chemical crosslinking. Cell lysates prepared from HeLa (F) cells without chemical crosslinking were used for co-immunoprecipitation (IP) with Protein G beads bound to rabbit normal IgG (negative control) or rabbit anti-hFis1 antibody as indicated, followed by Western blotting with indicated antibodies.


G , H . Interaction of hFis1 with Mfn1 / 2 with OPA1 are independent events . WT 293T were treated with control , OPA1 ( G ) or Mfn1 plus Mfn2 ( H ) siRNA , followed by in vivo crosslinking with 1 % FA . lysates were used for co - IP with G beads bound to rabbit normal IgG ( negative control ) or rabbit anti - hFis1 antibody as indicated , followed by immunoblotting with indicated antibodies .

O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O B-MEASURED_VAR O O O O O O O O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O

G, H. Interaction of hFis1 with Mfn1/2 and with OPA1 are independent events. WT 293T cells were treated with control, OPA1 (G) or Mfn1 plus Mfn2 (H) siRNA, followed by in vivo crosslinking with 1% FA. Cell lysates were used for co-IP with Protein G beads bound to rabbit normal IgG (negative control) or rabbit anti-hFis1 antibody as indicated, followed by immunoblotting with indicated antibodies.


I . Interactions between Mfn1 / 2 OPA1 occur independent of hFis1 . WT 293T were treated with control or hFis1 siRNA , followed by in vivo crosslinking with 1 % FA . lysates were used for co - IP with G beads bound to mouse normal IgG ( negative control ) or mouse anti - OPA1 antibody as indicated , followed by immunoblotting with indicated antibodies .

O O O O B-MEASURED_VAR O O B-MEASURED_VAR O O O B-MEASURED_VAR O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O

I. Interactions between Mfn1/2 and OPA1 occur independent of hFis1. WT 293T cells were treated with control or hFis1 siRNA, followed by in vivo crosslinking with 1% FA. Cell lysates were used for co-IP with Protein G beads bound to mouse normal IgG (negative control) or mouse anti-OPA1 antibody as indicated, followed by immunoblotting with indicated antibodies.


J . Interaction between Mfn1 Mfn2 is not affected by hFis1 overexpression . 293T were transfected with empty vector or Myc - hFis1 , followed by in vivo crosslinking with 1 % FA . lysates were used for co - IP with G beads bound to mouse normal IgG or mouse anti - Mfn1 antibody , followed by immunoblotting with indicated antibodies .

O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O B-MEASURED_VAR O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O

J. Interaction between Mfn1 and Mfn2 is not affected by hFis1 overexpression. 293T cells were transfected with empty vector or Myc-hFis1, followed by in vivo crosslinking with 1% FA. Cell lysates were used for co-IP with Protein G beads bound to mouse normal IgG or mouse anti-Mfn1 antibody, followed by immunoblotting with indicated antibodies.


B . Interaction of different hFis1 mutants with Mfns OPA1 . hFis1 - / - 293T were transfected with full - truncated hFis1 mutants as indicated , followed by in vivo crosslinking with 1 % FA . lysates were used for co - IP with anti - Myc or anti - GFP agarose beads immunoprecipitates were analyzed by Western blotting with indicated antibodies .

O O O O O B-MEASURED_VAR O O O B-MEASURED_VAR O B-CONTROLLED_VAR O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

B. Interaction of different hFis1 mutants with Mfns and OPA1. hFis1-/- 293T cells were transfected with the full-length and the truncated hFis1 mutants as indicated, followed by in vivo crosslinking with 1% FA. Cell lysates were used for co-IP with anti-Myc or anti-GFP agarose beads and the immunoprecipitates were analyzed by Western blotting with indicated antibodies.


A , B . Representative confocal of mitochondrial fusion in WT 293T polykaryons in cultures subjected to PEG - fusion in presence or absence of exogenous hFis1 . WT 293T stably expressing mitoGFP or mitoRFP were co - cultured , subsequently transfected with empty vector ( control ) or with Myc - hFis1 as indicated then treated with PEG for fusion . Mitochondrial fusion is indicated by co - localization of mitoGFP mitoRFP ( i . e . yellow mitochondria ) . Left panel : empty vector transfected ; right panel : Myc - hFis1transfected . Insets represent magnification of boxed areas in upper panel ( A ) . Quantitative of extent of mitochondrial fusion in hybrid was performed by Pearson ' s correlation coefficient ( PCC ) in three independent experiments for each condition data summarized in ( B ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A, B. Representative confocal images of mitochondrial fusion in WT 293T polykaryons in cultures subjected to the PEG-based fusion assay in the presence or absence of exogenous hFis1. WT 293T cells stably expressing mitoGFP or mitoRFP were co-cultured, subsequently transfected with empty vector (control) or with Myc-hFis1 as indicated and then treated with PEG for cell fusion. Mitochondrial fusion is indicated by co-localization of mitoGFP and mitoRFP (i.e. yellow mitochondria). Left panel: empty vector transfected; right panel: Myc-hFis1transfected. Insets represent magnification of the boxed areas in upper panel (A). Quantitative analysis of the extent of mitochondrial fusion in individual hybrid cells was performed by the Pearson's correlation coefficient (PCC) in three independent experiments for each condition and data summarized in (B).


C , D . Representative confocal of mitochondrial fusion in WT 293T assessed by mito - PAGFP - fusion in presence or absence of exogenous hFis1 . WT 293T co - transfected with mito - PAGFP ( 0 . 5 µg ) , mito - DsRed ( 0 . 2 µg ) , either empty vector ( 0 . 5 µg , upper panel ) or hFis1 ( 0 . 5 µg , lower panel ) were photoactivated in a small region of interest ( ROI ) ( circle , 3 µm diameter ) in preactivation of mitochondria seen by mito - DsRed ( red ) . After photoactivation , sequential Z - stack with photoactivated GFP ( green ) mitochondrial marker ( mito - DsRed ) were collected at indicated time points using series of Z - sections from top to bottom with intervals between sections set to 0 . 5 - 0 . 75 μm ( C ) . Mitochondrial fusion was quantified by analyzing changes in of photoactivated mito - PAGFP in ROIs at 40 sec , 15 , 30 , 45 min after photoactivation . dilution rates ( ) of GFP at different time points were normalized by at 40 sec after photoactivation ( D ) .

O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

C, D. Representative confocal images of mitochondrial fusion in WT 293T cells assessed by the mito-PAGFP-based fusion assay in the presence or absence of exogenous hFis1. WT 293T cells co-transfected with mito-PAGFP (0.5 µg), mito-DsRed (0.2 µg), and either empty vector (0.5 µg, the upper panel) or hFis1 (0.5 µg, the lower panel) were photoactivated in a small region of interest (ROI) (white circle, 3 µm diameter) in preactivation images of mitochondria seen by mito-DsRed (red). After photoactivation, sequential Z-stack images with photoactivated GFP (green) and mitochondrial marker (mito-DsRed) were collected at indicated time points using series of Z-sections from the top to the cell bottom with intervals between sections set to 0.5-0.75 μm (C). Mitochondrial fusion was quantified by analyzing changes in fluorescence intensity of photoactivated mito-PAGFP in ROIs at 40 sec, 15, 30, 45 min after photoactivation. The dilution rates (percentage) of the GFP fluorescence intensity at different time points were normalized by the fluorescence intensity at 40 sec after photoactivation (D).


E , F . Representative confocal of mitochondrial fusion in WT 293T polykaryons in cultures subjected to PEG - fusion in presence or absence of endogenous hFis1 . Both WT 293T stably expressing mitoGFP or mitoRFP were transfected with scrambled siRNA ( control siRNA ) or hFis1 - siRNA as indicated then co - cultured fused using PEG treatment . Left panel : control siRNA transfected ; right panel : hFis1 - siRNA transfected ( E ) . Quantitative of mitochondrial fusion in hybrid was performed by Pearson ' s correlation coefficient in three independent experiments data summarized in ( F ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

E, F. Representative confocal images of mitochondrial fusion in WT 293T polykaryons in cultures subjected to the PEG-based fusion assay in the presence or absence of endogenous hFis1. Both WT 293T cells stably expressing mitoGFP or mitoRFP were transfected with scrambled siRNA (control siRNA) or hFis1-siRNA as indicated and then co-cultured and fused using PEG treatment. Left panel: control siRNA transfected; right panel: hFis1-siRNA transfected (E). Quantitative analysis of the mitochondrial fusion in individual hybrid cells was performed by the Pearson's correlation coefficient in three independent experiments and data summarized in (F).


G , H . Representative confocal of mitochondrial fusion in WT 293T assessed by mito - PAGFP - fusion in presence or absence of endogenous hFis1 . Scrambled siRNA or hFis1 siRNA treated were co - transfected with mito - PAGFP mito - DsRed were subsequently photoactivated ( G ) as described in ( C ) . Mitochondrial fusion was quantified ( H ) as described in ( D ) .

O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

G, H. Representative confocal images of mitochondrial fusion in WT 293T cells assessed by the mito-PAGFP-based fusion assay in the presence or absence of endogenous hFis1. Scrambled siRNA or hFis1 siRNA treated cells were co-transfected with mito-PAGFP and mito-DsRed and were subsequently photoactivated (G) as described in (C). Mitochondrial fusion was quantified (H) as described in (D).


A , B . Representative confocal of mitochondrial fusion in Drp1 - / - 293T polykaryons in cultures subjected to PEG - fusion in presence or absence of exogenous hFis1 . Drp1 - / - stably expressing mitoGFP or mitoRFP were co - cultured , subsequently transfected with empty vector ( control ) or Myc - hFis1 as indicated then stimulated with PEG treatment for fusion , followed by immunostaining with anti - Myc antibody ( green ) . Mitochondrial fusion was indicated by co - localization of mitoGFP mitoRFP ( i . e . yellow mitochondria ) . Left panel : empty vector transfected ; right panel : Myc - hFis1 transfected . Insets represent magnification of boxed areas in upper panel ( A ) . Quantitative analyses for measuring extent of mitochondrial fusion in hybrid were performed by Pearson ' s correlation coefficient ( PCC ) in three independent experiments summarized in ( B ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A, B. Representative confocal images of mitochondrial fusion in Drp1-/- 293T polykaryons in cultures subjected to the PEG-based fusion assay in the presence or absence of exogenous hFis1. Drp1-/- cells stably expressing mitoGFP or mitoRFP were co-cultured, subsequently transfected with empty vector (control) or Myc-hFis1 as indicated and then stimulated with PEG treatment for cell fusion, followed by immunostaining with anti-Myc antibody (green). Mitochondrial fusion was indicated by co-localization of mitoGFP and mitoRFP (i.e. yellow mitochondria). Left panel: empty vector transfected; right panel: Myc-hFis1 transfected. Insets represent magnification of the boxed areas in upper panel (A). Quantitative analyses for measuring the extent of mitochondrial fusion in individual hybrid cells were performed by the Pearson's correlation coefficient (PCC) in three independent experiments and summarized in (B).


C , D . Representative confocal of mitochondrial fusion in Drp1 - / - 293T assessed by mito - PAGFP - fusion in presence or absence of exogenous hFis1 . Drp1 - / - co - transfected with mito - PAGFP , mito - DsRed , either empty vector ( upper panel ) or hFis1 ( lower panel ) were photoactivated in a small region of interest ( ROI ) ( circle , 3 µm diameter ) as indicated in preactivation of mitochondria seen by mito - DsRed ( red ) . After photoactivation , Z - stack with photoactivated GFP ( green ) mitochondrial marker ( mito - DsRed ) were collected at times 40 sec , 15 , 30 , 45 min as indicated ( C ) . Mitochondrial fusion was quantified by of changes in of photoactivated mito - PAGFP in ROIs at 40 sec , 15 , 30 , 45 min after photoactivation . dilution rates ( ) of GFP at different time points were normalized by at 40 sec after photoactivation ( D ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

C, D. Representative confocal images of mitochondrial fusion in Drp1-/- 293T cells assessed by the mito-PAGFP-based fusion assay in the presence or absence of exogenous hFis1. Drp1-/- cells co-transfected with mito-PAGFP, mito-DsRed, and either empty vector (the upper panel) or hFis1 (the lower panel) were photoactivated in a small region of interest (ROI) (white circle, 3 µm diameter) as indicated in preactivation images of mitochondria seen by mito-DsRed (red). After photoactivation, Z-stack images with photoactivated GFP (green) and mitochondrial marker (mito-DsRed) were collected at times 40 sec, 15, 30, 45 min as indicated (C). Mitochondrial fusion was quantified by analysis of changes in the fluorescence intensity of photoactivated mito-PAGFP in ROIs at 40 sec, 15, 30, 45 min after photoactivation. The dilution rates (percentage) of the GFP fluorescence intensity at different time points were normalized by the fluorescence intensity at 40 sec after photoactivation (D).


E , F . Representative confocal of mitochondrial fusion in Drp1 - / - 293T polykaryons in cultures subjected to PEG - fusion in presence or absence of endogenous hFis1 . Drp1 - / - stably expressing mitoGFP or mitoRFP were transfected with scrambled siRNA ( control siRNA ) or with hFis1 - siRNA as indicated then co - cultured fused by PEG treatment . Left panel : control siRNA transfected ; right panel : hFis1 - siRNA transfected ( E ) . Quantitative of extent of mitochondrial fusion in hybrid was performed by Pearson ' s correlation coefficient ( PCC ) in three independent experiments summarized in ( F ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

E, F. Representative confocal images of mitochondrial fusion in Drp1-/- 293T polykaryons in cultures subjected to the PEG-based fusion assay in the presence or absence of endogenous hFis1. Drp1-/- cells stably expressing mitoGFP or mitoRFP were transfected with scrambled siRNA (control siRNA) or with hFis1-siRNA as indicated and then co-cultured and fused by PEG treatment. Left panel: control siRNA transfected; right panel: hFis1-siRNA transfected (E). Quantitative analysis of extent of mitochondrial fusion in individual hybrid cells was performed by the Pearson's correlation coefficient (PCC) in three independent experiments and summarized in (F).


G , H . Representative confocal of mitochondrial fusion in Drp1 - / - 293T monitored by mito - PAGFP - fusion in presence or absence of endogenous hFis1 . Scrambled siRNA or hFis1 siRNA treated were co - transfected with mito - PAGFP mito - DsRed were subsequently photoactivated ( G ) as described in ( C ) . Mitochondrial fusion was quantified by of changes in of photoactivated mito - PAGFP ( H ) as described in ( D ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

G, H. Representative confocal images of mitochondrial fusion in Drp1-/- 293T cells monitored by the mito-PAGFP-based fusion assay in the presence or absence of endogenous hFis1. Scrambled siRNA or hFis1 siRNA treated cells were co-transfected with mito-PAGFP and mito-DsRed and were subsequently photoactivated (G) as described in (C). Mitochondrial fusion was quantified by analysis of changes in the fluorescence intensity of photoactivated mito-PAGFP (H) as described in (D).


A , B . GTP hydrolysis activities of Mfn1 , Mfn2 , Mfn2K109A OPA1 in presence or absence of recombinant GST - hFis1 or GST ( negative control ) were determined by measuring of free phosphate ( Pi ) released from GTP . Myc - tagged Mfn1 , Mfn2 , Mfn2K109A OPA1 were transiently expressed separately in 293T immunopurified by anti - Myc agarose beads . GTPase activities of immunopurified Myc - tagged Mfn1 , Mfn2 , Mfn2K109A OPA1 were determined after treatment with or without recombinant GST - hFis1 or GST ( A ) . input of immunopurified Mfn1 - Myc , Mfn2 - Myc OPA1 - Myc ( left panel ) as well as recombinant GST - hFis1 ( ~ 42 kDa ) GST ( ~ 26 kDa ) ( right panel ) were assessed by immunoblotting with indicated antibodies ( B ) .

O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O B-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O B-MEASURED_VAR O O O B-MEASURED_VAR O O B-MEASURED_VAR O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O

A, B. GTP hydrolysis activities of Mfn1, Mfn2, Mfn2K109A and OPA1 in the presence or absence of recombinant GST-hFis1 or GST (negative control) were determined by measuring the concentration of free phosphate (Pi) released from GTP. Myc-tagged Mfn1, Mfn2, Mfn2K109A and OPA1 were transiently expressed separately in 293T cells and immunopurified by anti-Myc agarose beads. GTPase activities of immunopurified Myc-tagged Mfn1, Mfn2, Mfn2K109A and OPA1 were determined after treatment with or without recombinant GST-hFis1 or GST protein (A). The input levels of immunopurified Mfn1-Myc, Mfn2-Myc and OPA1-Myc (left panel) as well as recombinant GST-hFis1 (~42 kDa) and GST (~26 kDa) (right panel) were assessed by immunoblotting with indicated antibodies (B).


C , D . GTP hydrolysis activities of Drp1 in presence or absence of recombinant GST - hFis1 or GST ( negative control ) were determined as described in ( A ) . Untagged WT Drp1 Drp1Q34A were transiently expressed in Drp1 - / - 293T immunopurified by G beads pre - incubated with Drp1 antibody . GTPase activities of immunopurified Drp1 Drp1Q34A were determined after treatment with or without recombinant hFis1 or GST ( C ) . input of immunopurified Drp1 Drp1Q34A were assessed by immunoblotting with anti - Drp1 antibody ( D ) .

O O O O O O O O B-MEASURED_VAR O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O B-MEASURED_VAR O O O O O

C, D. GTP hydrolysis activities of Drp1 in the presence or absence of recombinant GST-hFis1 or GST (negative control) were determined as described in (A). Untagged WT Drp1 and Drp1Q34A were transiently expressed in Drp1-/- 293T cells and immunopurified by protein G beads pre-incubated with Drp1 antibody. GTPase activities of immunopurified Drp1 and Drp1Q34A were determined after treatment with or without recombinant hFis1 or GST protein (C). The input levels of immunopurified Drp1 and Drp1Q34A were assessed by immunoblotting with anti-Drp1 antibody (D).


E , F . GTP hydrolysis activities of Dyn2 in presence or absence of recombinant GST - hFis1 or GST ( negative control ) were determined as described in ( A ) . Endogenous Dyn2 in Drp1 - / - 293T was immunopurified by G beads pre - incubated with anti - Dyn2 antibody . GTPase activity of immunopurified Dyn2 was determined after treatment with or without recombinant GST - hFis1 or GST ( E ) . input of immunopurified Dyn2 were assessed by immunoblotting with anti - Dyn2 antibody ( F ) .

O O O O O O O O B-MEASURED_VAR O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O B-MEASURED_VAR O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O B-MEASURED_VAR O O O O O O O B-MEASURED_VAR O O O O O

E, F. GTP hydrolysis activities of Dyn2 in the presence or absence of recombinant GST-hFis1 or GST (negative control) were determined as described in (A). Endogenous Dyn2 in Drp1-/- 293T cells was immunopurified by protein G beads pre-incubated with anti-Dyn2 antibody. GTPase activity of immunopurified Dyn2 was determined after treatment with or without recombinant GST-hFis1 or GST protein (E). The input levels of immunopurified Dyn2 were assessed by immunoblotting with anti-Dyn2 antibody (F).


G . Summary of effects of pro - fusion GTPases ( Mfns OPA1 ) on hFis1 - induced mitochondrial fragmentation in Drp1 - / - . Percentages ( mean ± s . e . m . ) of with indicated mitochondrial morphologies in Drp1 - / - 293T co - expressing Myc - hFis1 either empty vector , Mfn1 - Myc , Mfn2 - Myc or OPA1 - Myc in three independent experiments . n represents number of analyzed . Corresponding confocal shown in Fig EV4 .

O O O O O O O O O O O O B-CONTROLLED_VAR O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O

G. Summary of the effects of pro-fusion GTPases (Mfns and OPA1) on hFis1-induced mitochondrial fragmentation in Drp1-/- cells. Percentages (mean ± s.e.m.) of cells with indicated mitochondrial morphologies in Drp1-/- 293T cells co-expressing Myc-hFis1 and either empty vector, Mfn1-Myc, Mfn2-Myc or OPA1-Myc in three independent experiments. n represents the number of cells analyzed. Corresponding confocal images shown in Fig EV4.


A . Ablation of OPA1 using CRISPR - Cas9 gene editing in Drp1 - / - 293T was confirmed by Western blotting .

O O O O B-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O

A. Ablation of OPA1 using CRISPR-Cas9 gene editing in Drp1-/- 293T cells was confirmed by Western blotting analysis.


B . Confocal of mitochondrial in Drp1 - / - ( left panel ) Drp1 / OPA1DKO 293T ( right panel ) . ( Aggr / frag : aggregation / fragmentation of mitochondria ; Bulb / tubular : bulb - like / tubular mitochondria ) .

O O O O O O B-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

B. Confocal images of mitochondrial morphology in Drp1-/- (left panel) and Drp1/OPA1DKO 293T (right panel) cells. (Aggr/frag: aggregation/fragmentation of mitochondria; Bulb/tubular: bulb-like/tubular mitochondria).


C . Confocal of mitochondrial in Drp1 / OPA1DKO 293T treated with indicated siRNAs , combinations of siRNAs or with Myc - hFis1 plasmid ( lower three panels ) compared to mitochondrial in Drp1 - / - 293T overexpressing Myc - hFis1 or not ( upper panel ) .

O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O B-MEASURED_VAR O O O O O O O

C. Confocal images of mitochondrial morphology in Drp1/OPA1DKO 293T cells treated with the indicated siRNAs, combinations of siRNAs or with Myc-hFis1 plasmid (lower three panels) compared to mitochondrial morphology in Drp1-/- 293T cells overexpressing Myc-hFis1 or not (upper panel).


D . Percentages ( mean ± s . e . m . ) of with indicated mitochondrial morphologies in Drp1 - / - 293T transfected with empty vector or Myc - hFis1 plasmid , in Drp1 / OPA1DKO 293T treated with indicated siRNA , combinations of siRNA or Myc - hFis1 plasmid . data were collected from three independent experiments . Total numbers ( n ) used for statistical are indicated for each condition .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O

D. Percentages (mean ± s.e.m.) of cells with indicated mitochondrial morphologies in Drp1-/- 293T cells transfected with empty vector or Myc-hFis1 plasmid, and in Drp1/OPA1DKO 293T cells treated with the indicated siRNA, combinations of siRNA or Myc-hFis1 plasmid. The data were collected from three independent experiments. Total cell numbers (n) used for statistical analysis are indicated for each condition.


E . Knockdown of indicated by siRNA in Drp1 / OPA1DKO 293T was confirmed by Western blotting .

O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O

E. Knockdown of the indicated proteins by siRNA in Drp1/OPA1DKO 293T cells was confirmed by Western blotting analysis.


Expression of Mpc in mitochondria assessed by SDS - PAGE immunoblot of crude mitochondrial preparations . untagged Mpc1 ( Mpc1 ) , histidine - tagged Mpc1 ( Mpc1his ) , histidine - tagged Mpc3 ( Mpc3his ) or Mpc1 / Mpc3 hetero - ( Mpc1 / Mpc3his ) were detected with antibodies raised against Mpc1 ( left panel ) or Mpc3 ( right panel ) are shown with dashed arrows .

O O O O O O O O O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O B-MEASURED_VAR O B-MEASURED_VAR O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O B-MEASURED_VAR O O O O O B-MEASURED_VAR O O O O O O O O O O

Expression of Mpc proteins in mitochondria assessed by SDS-PAGE and immunoblot analysis of crude mitochondrial preparations. The individual untagged Mpc1 (Mpc1), histidine-tagged Mpc1 (Mpc1his), histidine-tagged Mpc3 (Mpc3his) or the Mpc1/Mpc3 hetero-complex (Mpc1/Mpc3his) were detected with antibodies raised against Mpc1 (left panel) or Mpc3 (right panel) and are shown with dashed arrows.


Five micrograms of each affinity - purified Mpc were analysed by SDS - PAGE bands were visualised by Coomassie Blue stain . Peptide mass finger printing was used to identify major

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Five micrograms of each affinity-purified Mpc protein were analysed by SDS-PAGE and the bands were visualised by Coomassie Blue stain. Peptide mass finger printing was used to identify the major protein bands


stability of purified was assessed via thermal denaturation by CPM - adduct formation . thermal denaturation profiles ( left ) were used to calculate first derivative ( right ) , which provides apparent melting , indicated with same colour coding .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

The stability of the purified proteins was assessed via thermal denaturation by fluorescent CPM-adduct formation. The thermal denaturation profiles (left) were used to calculate the first derivative (right), which provides the apparent melting temperature, indicated with the same colour coding.


stability of same samples , as in panel ( D ) , was assessed by NanoDSF . changes in 330 nm / 350 nm with ( left ) were used to calculate first derivative ( right ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

The stability of the same samples, as in panel (D), was assessed by NanoDSF. The changes in the 330 nm/350 nm ratio with temperature (left) were used to calculate the first derivative (right).


Nickel - affinity - purified Mpc1 / Mpc3 hetero - used for SEC - MALLS , showing a 1 : 1 stoichiometry of protomers .

O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O O O

Nickel-affinity-purified Mpc1/Mpc3 hetero-complex used for SEC-MALLS analysis, showing a 1:1 stoichiometry of the protomers.


SEC - MALLS of hetero - . scattering trace for Mpc1 / Mpc3 is shown as a black . masses of - detergent - lipid ( PDL , green ) , detergent - lipid micelle ( DL , blue ) ( P , red ) are indicated . fractions across peak were assessed by SDS - PAGE visualised by Coomassie Blue ( B , inset ) .

O O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

SEC-MALLS analysis of the hetero-complex. The light scattering trace for Mpc1/Mpc3 is shown as a black line. The masses of the protein-detergent-lipid complex (PDL, green), the detergent-lipid micelle (DL, blue) and the protein (P, red) are indicated. Protein fractions across the peak were assessed by SDS-PAGE and visualised by Coomassie Blue staining (B, inset).


Nickel - affinity - purified Mpc3 used for SEC - MALLS .

Nickel-affinity-purified Mpc3 protein used for SEC-MALLS analysis.


SEC - MALLS of Mpc3 , colour designation as in ( B ) . fractions across peak ( D , inset ) were assessed as in ( B ) .

O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O

SEC-MALLS analysis of Mpc3, colour designation as in (B). The protein fractions across the peak (D, inset) were assessed as in (B).


Time course of pyruvate homo - exchange by Mpc1 / Mpc3 hetero - in liposomes ( n = 8 ) in comparison to empty liposomes ( n = 6 ) at a ΔpH of 1 . 6 .

O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O

Time course of pyruvate homo-exchange by the Mpc1/Mpc3 hetero-complex in liposomes (n=8) in comparison to empty liposomes (n=6) at a ΔpH of 1.6.


Kinetic of pyruvate homo - exchange at ΔpH of 1 . 6 ( n = 3 ) . hetero - was assayed for initial rates of uptake in range of 25 - 600 μM . calculated KΜ was 299 μΜ in this experiment 318 409 μΜ in two additional biological repeats .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Kinetic analysis of pyruvate homo-exchange at ΔpH of 1.6 (n=3). The hetero-complex was assayed for initial rates of uptake in the concentration range of 25-600 μM. The calculated KΜ was 299 μΜ in this experiment and 318 and 409 μΜ in two additional biological repeats.


Time course of pyruvate homo - exchange by Mpc1 / Mpc3 hetero - in physiological pH ( n = 4 ) compared to empty liposomes ( n = 4 ) .

O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O

Time course of pyruvate homo-exchange by the Mpc1/Mpc3 hetero-complex in physiological pH (n=4) compared to empty liposomes (n=4).


In absence of a ΔpH , time course of pyruvate homo - exchange was similar for Mpc1 / Mpc3 proteoliposomes empty liposomes ( n = 4 ) .

O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O

In the absence of a ΔpH, the time course of pyruvate homo-exchange was similar for the Mpc1/Mpc3 proteoliposomes and the empty liposomes (n=4).


Inhibition of [ 14C ] - pyruvate homo - exchange by UK5099 ( 1 - 100 μM ) , Zaprinast ( 1 - 1000 μM ) , lonidamine ( 10 - 10000 μM ) 7ACC2 ( 5 - 500 μM ) . Data points represent mean of three technical replicates of a typical experiment . IC50 measurements have also been independently replicated , three times for UK5099 Zaprinast 7ACC2 ( average IC50 9 ± 7 μM , 18 ± 8 μM 27 ± 13 μM , respectively ) two times for lonidamine ( average IC50 , 118 ± 24 μM ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Inhibition of [14C]-pyruvate homo-exchange by UK5099 (1-100 μM), Zaprinast (1-1000 μM), lonidamine (10-10000 μM) and 7ACC2 (5-500 μM). Data points represent the mean of three technical replicates of a typical experiment. The IC50 measurements have also been independently replicated, three times for UK5099 Zaprinast and 7ACC2 (average IC50 9 ± 7 μM, 18 ± 8 μM and 27 ± 13 μM, respectively) and two times for lonidamine (average IC50, 118 ± 24 μM).


[ 14C ] - pyruvate homo - exchange inhibition by TZDs , pioglitazone rosiglitazone ( n = 6 ) .

[14C]-pyruvate homo-exchange inhibition by the TZDs, pioglitazone and rosiglitazone (n=6).


Time course of pyruvate homo - exchange in proteoliposomes at a ΔpH of 1 . 6 was compared for Mpc3 ( n = 4 ) , Mpc1 / Mpc3 hetero - ( n = 6 ) empty liposomes ( n = 4 ) .

O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O

Time course of pyruvate homo-exchange in proteoliposomes at a ΔpH of 1.6 was compared for Mpc3 (n=4), the Mpc1/Mpc3 hetero-complex (n=6) and empty liposomes (n=4).


Time course of pyruvate homo - exchange at a ΔpH of 1 . 6 was compared for Mpc1 ( n = 4 ) , Mpc1 / Mpc3 hetero - ( n = 4 ) empty liposomes ( n = 4 ) .

O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O

Time course of pyruvate homo-exchange at a ΔpH of 1.6 was compared for Mpc1 (n=4), Mpc1/Mpc3 hetero-complex (n=4) and empty liposomes (n=4).


Thermostability via cpm . In detergent solution , Zaprinast ( 250 μM ) increased thermostability of Mpc1 / Mpc3 hetero - ( black lines ) ( n = 3 ) but not of Mpc3 ( blue lines ) ( n = 3 ) .

O O O O O O O O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O

Thermostability analysis via cpm. In detergent solution, Zaprinast (250 μM) increased the thermostability of the Mpc1/Mpc3 hetero-complex (black lines) (n=3) but not of Mpc3 (blue lines) (n=3).


Thermostability via nanoDSF . In detergent solution , Zaprinast ( 250 μM ) increased thermostability of Mpc1 / Mpc3 hetero - ( black lines ) ( n = 3 ) but not of Mpc1 ( orange lines ) ( n = 3 ) .

Thermostability analysis via nanoDSF. In detergent solution, Zaprinast (250 μM) increased the thermostability of the Mpc1/Mpc3 hetero-complex (black lines) (n=3) but not of Mpc1 (orange lines) (n=3).


was observed in transformed N . benthamiana leaf epidermal , which results from complementation of N - terminal part of YFP fused with ATG8a ( ATG8a - N - YFP ) by C - terminal part of YFP fused with NBR1 ( NBR1 - C - YFP ) . No was observed when ATG8a - N - YFP was coexpressed with unfused C - YFP or with mNBR1 - C - YFP or when unfused N - YFP was coexpressed with NBR1 - C - YFP . YFP epifluorescence , bright - field overlay of same are shown .

O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O B-MEASURED_VAR O B-CONTROLLED_VAR O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O

Fluorescence was observed in the transformed N. benthamiana leaf epidermal cells, which results from complementation of the N-terminal part of the YFP fused with ATG8a (ATG8a-N-YFP) by the C-terminal part of the YFP fused with NBR1 (NBR1-C-YFP). No fluorescence was observed when ATG8a-N-YFP was coexpressed with unfused C-YFP or with mNBR1-C-YFP or when unfused N-YFP was coexpressed with NBR1-C-YFP. YFP epifluorescence images, bright-field images and overlay images of the same cells are shown.


Five - old Arabidopsis wild - type Col - 0 were placed in 22 ° C 45 ° C growth chambers total was isolated from leaf samples collected at indicated times . Transcript were determined using real - time qRT - PCR . Error bars indicate SE ( n = 3 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Five weeks-old Arabidopsis wild-type Col-0 plants were placed in the 22°C and 45°C growth chambers and total RNA was isolated from leaf samples collected at indicated times. Transcript levels were determined using real-time qRT-PCR. Error bars indicate SE (n = 3).


( A ) Four - old transgenic wild - type Col - 0 ( WT ) atg7 - 2 mutant expressing GFP - ATG8a were treated with ( 45 ° C ) or without ( 22 ° C ) heat shock for 3 h then placed at room for 0 . 5 h . leaves were visualized by confocal microscopy of GFP signal . ( B ) Numbers of punctate GFP - ATG8a spots representing autophagosomes per 10 , 000 µm2 section . Means SE were calculated from three experiments . According to Duncan ' s multiple range ( P = 0 . 05 ) , means do not differ significantly if they are indicated with same letter . Bar = 20 µm .

O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A) Four-weeks old transgenic wild-type Col-0 (WT) and atg7-2 mutant plants expressing GFP-ATG8a were treated with (45°C) or without (22°C) heat shock for 3 h and then placed at room temperature for 0.5 h. The leaves were visualized by fluorescence confocal microscopy of GFP signal. (B) Numbers of punctate GFP-ATG8a spots representing autophagosomes per 10,000 µm2 section. Means and SE were calculated from three experiments. According to Duncan's multiple range test (P = 0.05), means do not differ significantly if they are indicated with the same letter. Bar = 20 µm.


( Top panel ) Four - old transgenic wild - type Col - 0 ( WT ) , atg7 - 2 nbr1 - 1 mutant expressing GFP - NBR1 were treated with ( 45 ° C ) or without ( 22 ° C ) heat shock for 3 h then placed at room for 0 . 5 h . leaves were visualized by confocal microscopy of GFP signal . ( Bottom panel ) Numbers of punctate GFP - NBR1 spots per 10 , 000 µm2 section . Means SE were calculated from three experiments . According to Duncan ' s multiple range ( P = 0 . 05 ) , means do not differ significantly if they are indicated with same letter . Bar = 20 µm .

O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O B-CONTROLLED_VAR O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(Top panel) Four-weeks old transgenic wild-type Col-0 (WT), atg7-2 and nbr1-1 mutant plants expressing GFP-NBR1 were treated with (45°C) or without (22°C) heat shock for 3 h and then placed at room temperature for 0.5 h. The leaves were visualized by fluorescence confocal microscopy of GFP signal. (Bottom panel) Numbers of punctate GFP-NBR1 spots per 10,000 µm2 section. Means and SE were calculated from three experiments. According to Duncan's multiple range test (P = 0.05), means do not differ significantly if they are indicated with the same letter. Bar = 20 µm.


( A ) Five - old Arabidopsis Col - 0 wild type ( WT ) , atg5 , atg7 nbr1 mutant were placed in 22 ° C 45 ° C growth for 10 hours then moved to room for 3 - day recovery or ( B ) sprayed with 20 µM methyl viologen ( MV ) kept under for two days before picture of representative was taken . experiments were repeated three times with similar results .

O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A) Five weeks-old Arabidopsis Col-0 wild type (WT), atg5, atg7 and nbr1 mutant plants were placed in 22°C and 45°C growth for 10 hours and then moved to room temperature for 3-day recovery or (B) sprayed with 20 µM methyl viologen (MV) and kept under light for two days before the picture of representative plants was taken. The experiments were repeated three times with similar results.


( B ) Fv / Fm . ( C ) Average values for Fv / Fm . Five - old Arabidopsis Col - 0 wild type ( WT ) , atg5 , atg7 nbr1 mutant were placed in 22 ° C 45 ° C growth chambers . Fully expanded leaves were sampled after 10 hours in indicated temperatures immediately measured for electrolyte leakage Fv / Fm . Error bars in ( A ) ( C ) indicate SE ( n = 5 ) . According to Duncan ' s multiple range ( P = 0 . 05 ) , means of EL or Fv / Fm do not differ significantly if they are indicated with same letter . experiments were repeated twice with similar results .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(B) Fv/Fm images. (C) Average values for the Fv/Fm images. Five weeks-old Arabidopsis Col-0 wild type (WT), atg5, atg7 and nbr1 mutant plants were placed in 22°C and 45°C growth chambers. Fully expanded leaves were sampled after 10 hours in the indicated temperatures and immediately measured for electrolyte leakage and Fv/Fm. Error bars in (A) and (C) indicate SE (n = 5). According to Duncan's multiple range test (P = 0.05), means of EL or Fv/Fm do not differ significantly if they are indicated with the same letter. The experiments were repeated twice with similar results.


( A ) Five - old Arabidopsis Col - 0 wild type ( WT ) , atg5 , atg7 nbr1 mutant were placed into a growth chamber with approximately 50 % humidity . photograph of representative was taken 10 days after withholding watering . experiment was repeated twice with similar results .

O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A) Five weeks-old Arabidopsis Col-0 wild type (WT), atg5, atg7 and nbr1 mutant plants were placed into a growth chamber with approximately 50% humidity. The photograph of representative plants was taken 10 days after withholding watering. The experiment was repeated twice with similar results.


( B ) Seven days - old seedlings Col - 0 WT , atg5 , atg7 nbr1 grown on solid MS medium were transferred to same medium ( control ) or same medium containing 0 . 16 M NaCl photographed 5 days later . survived seedlings were scored 5 days after transfer average values SE were calculated from three experiments . According to Duncan ' s multiple range ( P = 0 . 05 ) , means do not differ significantly if they are indicated with same letter .

O O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(B) Seven days-old seedlings Col-0 WT, atg5, atg7 and nbr1 grown on solid MS medium were transferred to the same medium (control) or the same medium containing 0.16 M NaCl and photographed 5 days later. The survived seedlings were scored 5 days after the transfer and the average values and SE were calculated from three experiments. According to Duncan's multiple range test (P = 0.05), means do not differ significantly if they are indicated with the same letter.


( A ) Eight old wild type ( WT ) , atg5 , atg7 nbr1 grown under normal growth conditions in a growth chamber . ( B ) Four old WT , atg5 , atg7 nbr1 after being kept in for 6 days . ( C ) of that survived various lengths in as determined by resumption of growth . Each point represents average of 10 . experiments were repeated twice with similar results .

O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A) Eight weeks old wild type (WT), atg5, atg7 and nbr1 plants grown under normal growth conditions in a growth chamber. (B) Four weeks old WT, atg5, atg7 and nbr1 plants after being kept in the dark for 6 days. (C) Percentage of plants that survived the various lengths in dark as determined by resumption of growth. Each point represents the average of 10 plants. The experiments were repeated twice with similar results.


( A ) Responses of Col - 0 wild type ( WT ) , atg5 , atg7 nbr1 to Botrytis . Wild - type mutant were inoculated by spraying with spore suspension at a density of 2 . 5 × 105 spores ml − 1 , kept at high humidity . Pictures of representative were taken at 4 5 days post inoculation ( dpi ) . ( B ) Quantitative real - time PCR of B . cinerea ActA ( BcActA ) transcript in infected Arabidopsis at 5 dpi . experiments were repeated twice with similar results .

O O O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O

(A) Responses of Col-0 wild type (WT), atg5, atg7 and nbr1 plants to Botrytis. Wild-type and mutant plants were inoculated by spraying with spore suspension at a density of 2.5×105 spores ml−1, and kept at high humidity. Pictures of representative plants were taken at 4 and 5 days post inoculation (dpi). (B) Quantitative real-time PCR analysis of the B. cinerea ActA (BcActA) transcript levels in infected Arabidopsis plants at 5 dpi. The experiments were repeated twice with similar results.


( A ) Diagrams of wild - type mutant NBR1 . conserved W I residues in LIR motif between two UBA domains were changed to A residues in NBR1W661A / I664A ( mNBR1 ) mutant . ( B ) Five - old Arabidopsis Col - 0 wild type ( WT ) , nbr1 transgenic nbr1 expressing wild - type NBR1 ( lines 3 6 ; see Figure S2 ) or NBR1W661A / I664A ( mNBR1 ) ( lines 2 6 ; see Figure S2 ) mutant gene were placed in a 45 ° C growth chambers for 10 hours then moved to room for 3 - day recovery before picture as taken . experiment was repeated three times with similar results .

O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A) Diagrams of wild-type and mutant NBR1 proteins. The conserved W and I residues in the LIR motif between the two UBA domains were changed to A residues in the NBR1W661A/I664A (mNBR1) mutant protein. (B) Five weeks-old Arabidopsis Col-0 wild type (WT), nbr1 and transgenic nbr1 plants expressing the wild-type NBR1 (lines 3 and 6; see Figure S2) or the NBR1W661A/I664A (mNBR1) (lines 2 and 6; see Figure S2) mutant gene were placed in a 45°C growth chambers for 10 hours and then moved to room temperature for 3-day recovery before the picture as taken. The experiment was repeated three times with similar results.