( G ) In vivo pRBC uptake by cDC1 ( XCR1 + CD172a - ) cDC2 ( XCR1 - CD172a + ) analyzed at 6 days post - infection with PbA . GFP . Gates were positioned on auto - of cDC subset in day 6 PbA - infected mice . As GFP + events are rare , 107 events were acquired for each mouse . Symbols show mean + / - ( N = 4 mice per group ) of GFP + in each DC subset . * , P = 0 . 0005 by unpaired t - without assuming consistent SD .

O O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR 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 O O O O O O 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) In vivo pRBC uptake by cDC1 (XCR1+ CD172a-) and cDC2 (XCR1- CD172a+) analyzed at 6 days post-infection with PbA.GFP. Gates were positioned based on the auto-fluorescence of the cDC subset in day 6 PbA-infected mice. As GFP+ events are rare, 107 events were acquired for each mouse. Symbols show the mean percentage +/- SEM (N = 4 mice per group) of GFP+ cells in each DC subset. *, P = 0.0005 by unpaired t-test without assuming consistent SD.


( H , I ) I - Ab presentation by cDC isolated from naïve mice . ( H ) I - Ab - NL17 presentation by DC incubated with PbA pRBC at a of 5 : 1 , assessed with BEZ hybridomas . H , I : representative of 2 independent experiments with 20 naïve mice pooled per experiment . Dotted in D , E , F , H , I shows basal response of hybridomas in absence of antigen .

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

(H,I) I-Ab presentation by cDC isolated from naïve mice. (H) I-Ab-NL17 presentation by DC incubated with PbA pRBC at a ratio of 5:1, assessed with the BEZ hybridomas. H,I: representative of 2 independent experiments with 20 naïve mice pooled per experiment. Dotted line in D,E,F,H,I shows the basal response of the hybridomas in the absence of antigen.


( H , I ) I - Ab presentation by cDC isolated from naïve mice . ( I ) I - Ab - IR17 presentation by DC incubated with OVA - expressing E . coli at a of 20 : 1 , assessed with OTIIZ hybridomas . H , I : representative of 2 independent experiments with 20 naïve mice pooled per experiment . Dotted in D , E , F , H , I shows basal response of hybridomas in absence of antigen .

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

(H,I) I-Ab presentation by cDC isolated from naïve mice. (I) I-Ab-IR17 presentation by DC incubated with OVA-expressing E. coli at a ratio of 20:1, assessed with the OTIIZ hybridomas. H,I: representative of 2 independent experiments with 20 naïve mice pooled per experiment. Dotted line in D,E,F,H,I shows the basal response of the hybridomas in the absence of antigen.


( J ) Fold - change surface expression of I - Ab in cDC2 over cDC1 in naïve mice ( mean + / - of 4 experiments ) . Difference is not significant on unpaired t - with Welch ' s correction .

O O O O O O 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

(J) Fold-change surface expression of I-Ab in cDC2 over cDC1 in naïve mice (mean +/- SEM of 4 experiments). Difference is not significant based on unpaired t-test with Welch's correction.


( B ) Gating strategy to assess efficiency of cDC1 depletion .

(B) Gating strategy to assess efficiency of cDC1 depletion.


( C ) ( mean + / - ) of CD8α + out of Lin - B220 - CD11c + MHC IIhi . * , P = 0 . 0043 by Mann - Whitney .

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

(C) Percentage (mean +/- SEM) of CD8α+ out of Lin- B220- CD11c+ MHC IIhi cells. *, P = 0.0043 by Mann-Whitney test.


( D ) FACS plots showing proportion of CD11a + CD49d + CD4 T that produce IFN TNF in response to MutuDC loaded with RBC , PbA pRBC or ETRAMP10 . 2 peptide .

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

(D) FACS plots showing the proportion of CD11a+ CD49d+ CD4 T cells that produce IFN and TNF in response to MutuDC loaded with RBC, PbA pRBC or ETRAMP10.2 peptide.


( E ) Proportion of CD11a + CD49d + CD4 T that co - produce IFN TNF ( mean + / - ) in response to MutuDC loaded with indicated antigen , isolated from Karma mice treated ( open circles ) or not ( black circles ) with DT . Asterisks show significant differences between no DT + DT group by multiple unpaired t - without assuming consistent SD . RBC , P = 0 . 18 ; GAPDH . 1 , P = 0 . 019 ; EF1a , 0 . 039 ; ETRAMP , P = 0 . 00054 ; OVA , P = 0 . 25 ; pRBC , P = 0 . 015 .

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

(E) Proportion of CD11a+ CD49d+ CD4 T cells that co-produce IFN and TNF (mean +/- SEM) in response to MutuDC loaded with the indicated antigen, isolated from Karma mice treated (open circles) or not (black circles) with DT. Asterisks show significant differences between no DT and + DT group by multiple unpaired t-tests without assuming consistent SD. RBC, P = 0.18 ; GAPDH.1, P = 0.019 ; EF1a, 0.039 ; ETRAMP, P = 0.00054 ; OVA, P = 0.25 ; pRBC, P = 0.015.


( F ) Absolute numbers of IFN + TNF + CD11a + CD49d + CD4 + T responding to indicated stimuli ( mean + / - ) . GAPDH . 1 , P = 0 . 0047 ; EF1a , 0 . 00039 ; ETRAMP , P = 0 . 00022 ; pRBC , P = 0 . 00019 by multiple unpaired t - without assuming consistent SD .

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 B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR 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

(F) Absolute numbers of IFN+ TNF+ CD11a+ CD49d+CD4+ T cells responding to the indicated stimuli (mean +/- SEM). GAPDH.1, P = 0.0047 ; EF1a, 0.00039 ; ETRAMP, P = 0.00022 ; pRBC, P = 0.00019 by multiple unpaired t-tests without assuming consistent SD.


( G ) Blood parasitemia ( mean + / - ) at day 4 6 pi in Karma mice treated ( open circles ) or not ( black circles ) with DT . Day 4 , P = 0 . 022 ; Day 6 , P = 0 . 77 by multiple unpaired t - without assuming consistent SD . C , E , F , G : No DT group , N = 5 mice ; + DT group , N = 6 mice . Representative of 3 independent experiments .

O O O O O O O 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

(G) Blood parasitemia (mean +/- SEM) at day 4 and 6 pi in Karma mice treated (open circles) or not (black circles) with DT. Day 4, P = 0.022 ; Day 6, P = 0.77 by multiple unpaired t-tests without assuming consistent SD. C,E,F,G: No DT group, N = 5 mice ; + DT group, N = 6 mice. Representative of 3 independent experiments.


( H ) Absolute numbers of CD4 + T producing IFN , TNF or IL - 10 , either individually or in combination , following restimulation with MutuDC + PbA pRBC ( mean + / - ) . These are called cytokine + in next panels . * , P = 0 . 0023 by unpaired t - without assuming consistent SD .

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

(H) Absolute numbers of CD4+ T cells producing IFN, TNF or IL-10, either individually or in combination, following restimulation with MutuDC+PbA pRBC (mean +/- SEM). These cells are called cytokine+ in the next panels. *, P = 0.0023 by unpaired t-test without assuming consistent SD.


( I ) Multifunctionality ( IFN , TNF , IL - 10 ) of cytokine + T responding to MutuDC loaded with PbA pRBC .

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

(I) Multifunctionality analysis (IFN, TNF, IL-10) of cytokine+ T cells responding to MutuDC loaded with PbA pRBC.


( J ) Proportion of IL - 10 + IFN - TNF - among cytokine + T , in response to MutuDC loaded with indicated antigen ( mean + / - ) . GAPDH . 1 , P = 0 . 32 ; EF1a , 0 . 16 ; ETRAMP , P = 8 . 5x10 - 5 ; OVA , P = 0 . 11 ; pRBC , P = 0 . 0015 by multiple unpaired t - without assuming consistent SD . H , I , J : N = 3 mice per group .

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

(J) Proportion of IL-10+ IFN- TNF- among cytokine+ T cells, in response to MutuDC loaded with the indicated antigen (mean +/- SEM). GAPDH.1, P = 0.32 ; EF1a, 0.16 ; ETRAMP, P = 8.5x10-5 ; OVA, P = 0.11 ; pRBC, P = 0.0015 by multiple unpaired t-tests without assuming consistent SD. H,I,J: N = 3 mice per group.


D . of oocytes from with MOS variants . day of oocyte retrieval was defined as day 0 . Polar bodies were indicated by black arrows . Scale bar = 20 µm .

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

D. The morphology of oocytes and embryos from female patients with MOS variants. The day of oocyte retrieval was defined as day 0. Polar bodies were indicated by black arrows. Scale bar=20 µm.


B . immunofluorescence show pERK1 / 2 ( red ) dynamics in immature ( GV , n = 5 ) , mature ( MII , n = 10 ) , fertilized oocytes from unidentified control ( n = 6 ) , as well as in mature oocytes from 1 carrying homozygous MOSAsn95Lys variants . pERK1 / 2 activation was inhibited after U0126 treatment ( n = 6 ) . FITC - α - tubulin ( Green ) DAPI ( blue ) were used for co - . 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 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-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

B. The immunofluorescence images show the pERK1/2 (red) dynamics in immature (GV, n=5), mature (MII, n=10), and fertilized oocytes from unidentified control patients (n=6), as well as in mature oocytes from patient 1 carrying homozygous MOSAsn95Lys variants. pERK1/2 activation was inhibited after U0126 treatment (n=6). FITC-α-tubulin (Green) and DAPI (blue) were used for co-staining. Scale bar=10 µm.


C . Western blot of MEK1 / 2 ERK1 / 2 activation after transfection of FLAG - tagged wild - type MOS identified MOS variants in HEK 293 .

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

C. Western blot analysis of the MEK1/2 and ERK1/2 activation after transfection of FLAG-tagged wild-type MOS and identified MOS variants in HEK 293 cells.


F . Immunofluorescence of FLAG ( green ) pERK1 / 2 ( grey ) in oocytes after injection of different MOS variant mRNAs combined with mCherry mRNAs . signal of mCherry was directly captured after fixation , with DAPI for visualization ( blue ) . ( n = over 30 oocytes in each group ) . Scale bar = 10 µm .

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

F. Immunofluorescence of FLAG (green) and pERK1/2 (grey) in oocytes after injection of different MOS variant mRNAs combined with mCherry mRNAs. The signal of mCherry was directly captured after fixation, with DAPI staining for DNA visualization (blue). (n=over 30 oocytes in each group). Scale bar=10 µm.


A . Immunofluorescence of FLAG ( red ) F - actin ( gray ) in oocytes after microinjection of negative control or mouse Mos siRNAs combined with or without wild - type human MOS or MOS variants mRNAs culture in medium with 2 . 5 µM milrinone for 24 h , followed by release to maturation ( n = 15 - 20 oocytes each group ) . 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 B-CONTROLLED_VAR 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 O O O O 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. Immunofluorescence of FLAG (red) and F-actin (gray) in oocytes after microinjection of negative control or mouse Mos siRNAs combined with or without wild-type human MOS or MOS variants mRNAs and culture in medium with 2.5 µM milrinone for 24 h, followed by release to maturation (n=15-20 oocytes each group). Scale bar= 10 µm.


C . immunofluorescence of F - actin ( red ) TPX2 ( green ) in WT Erk1 / 2oo - / - oocytes , using DAPI ( blue ) co - ( n = 15 - 20 oocytes for each group ) . zoomed of F - actin are displayed in gray . Scale bar = 10 µm . D . box plot summarizing relative intensities of F - actin between control ERK1 / 2oo - / - oocytes ( n = 7 - 10 oocytes for each group ) .

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

C. The immunofluorescence of F-actin (red) and TPX2 (green) in WT and Erk1/2oo-/- oocytes, using DAPI (blue) co-staining (n=15-20 oocytes for each group). The zoomed images of F-actin are displayed in gray. Scale bar=10 µm. D. The box plot summarizing the relative intensities of F-actin between control and ERK1/2oo-/- oocytes (n=7-10 oocytes for each group).


E . Representative bright field showing of oocytes from wildtype ERK1 / 2oo - / - mice . MII oocyte , zygote , 2 - blastocyst were harvested in vivo at 14 , 24 , 48 , 96 h post - hCG , respectively ( n = 3 mice for each timepoints in two groups ) . Scale bar = 100 µm . F , G . Bar graphs shows blastocyst ( F ) fragment ( G ) in wild - type ( n = 51 ) maternal ERK1 / 2 deletion ( n = 63 ) .

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

E. Representative bright field images showing the morphology of oocytes and embryos from wildtype and ERK1/2oo-/- mice. The MII oocyte, zygote, 2-cell and blastocyst embryos were harvested in vivo at 14, 24, 48, and 96 h post-hCG, respectively (n=3 mice for each timepoints in two groups). Scale bar=100 µm. F, G. Bar graphs shows the blastocyst percentage (F) and fragment percentage (G) in wild-type (n=51) and maternal ERK1/2 deletion embryos (n=63).


H . bar graphs show expression of selected MOS - regulated decay genes ( n = 2 or 3 biological replicates ) .

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

H. The bar graphs show the expression levels of selected MOS-regulated decay genes (n=2 or 3 biological replicates).


H . showing expression level of selected maternal genes , mitochondrial translation genes mitochondrial respiratory chain genes regulated by MOS mutation or ERK1 / 2 inactivation .

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 O

H. Heatmap showing the expression level of selected maternal genes, mitochondrial translation genes and mitochondrial respiratory chain genes regulated by MOS mutation or ERK1/2 inactivation.


A . scatter plots showing ATP of human MII oocyte matured in medium with DMSO ( control , Ctrl , n = 11 ) or 20 uM U0126 treatment ( n = 12 ) . B . scatter plots showing ATP of mouse MII oocytes after in vitro maturation with DMSO ( control , Ctrl , n = 19 ) or 20 uM U0126 ( n = 17 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. The scatter plots showing the ATP levels of human MII oocyte matured in medium with DMSO (control, Ctrl, n=11) or 20 uM U0126 treatment (n=12). B. The scatter plots showing the ATP levels of mouse MII oocytes after in vitro maturation with DMSO (control, Ctrl, n=19) or 20 uM U0126 (n=17). C


C . representative showing JC - 1 monomers ( green ) ( red ) of mouse oocytes after treatment with DMSO ( control , Ctrl , n = 34 ) or U0126 ( n = 40 ) in in vitro culture medium for 16 h . Scale bar = 100 µm . D . bar graph of of intensities of JC - 1 to those of monomers in ( C ) . intensities of JC - 1 were quantified by Image J software . oocytes number used was labeled .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. The representative images showing the JC-1 monomers (green) and aggregates (red) of mouse oocytes after treatment with DMSO (control, Ctrl, n=34) or U0126 (n=40) in in vitro culture medium for 16 h. Scale bar=100 µm. D. The bar graph of ratio of intensities of JC-1 aggregates to those of monomers in (C). The intensities of JC-1 were quantified by Image J software. The oocytes number used was labeled. E


E . Representative displaying JC - 1 monomers ( green ) , ( red ) bright filed of GV MII oocytes from wild - type ( WT ) Erk1 / 2oo - / - oocytes ( n = 3 mice in each group ) . Erk1 / 2oo - / - oocytes exhibited big multiple polar bodies . Scale bar = 100 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 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

E. Representative images displaying JC-1 monomers (green), aggregates (red) and bright filed images of GV and MII oocytes from wild-type (WT) and Erk1/2oo-/- oocytes (n=3 mice in each group). Erk1/2oo-/- oocytes exhibited big and multiple polar bodies. Scale bar=100 µm.


F . Confocal of JC - 1 monomers ( green ) ( red ) of GV MII oocytes from wild - type ( WT ) Erk1 / 2oo - / - oocytes ( n = 3 mice in each group ) . Erk1 / 2oo - / - oocytes exhibited big multiple polar bodies , indicated by red arrows . Hoechst 33342 ( blue ) was co - stained to represent nucleus or chromosome . 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 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 O O

F. Confocal images of JC-1 monomers (green) and aggregates (red) of GV and MII oocytes from wild-type (WT) and Erk1/2oo-/- oocytes (n=3 mice in each group). Erk1/2oo-/- oocytes exhibited big and multiple polar bodies, indicated by red arrows. Hoechst 33342 (blue) was co-stained to represent nucleus or chromosome. Scale bar=10 µm.


( A Representative confocal of HeLa TRIM16KO treated with ( A ) MG132 ( 20 µM , 2h ) , scale bar : 7 . 5 µm . samples were processed for IF with Ub p62 antibody . ( B Representative confocal of control siRNA TRIM16 siRNA transfected treated with ( B ) MG132 ( 20 µM , 2h ) , where IF was conducted with Ub p62 antibodies . ( C graph shows of with Ub - p62 co - localized dots . Data from ≥ 10 fields ( 40X ) , n = 3 , Mean ± SD , * * p < 0 . 0003 , * p < 0 . 002 ( Student ' s unpaired t - ) . Data information : Unless otherwise stated , scale bar : 10 µm .

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 B-MEASURED_VAR 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-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 O O O O O O O O O O O O O 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 Representative confocal images of HeLa and TRIM16KO cells treated with (A) MG132 (20 µM, 2h), scale bar: 7.5 µm. and the samples were processed for IF analysis with Ub and p62 antibody. (B Representative confocal images of control siRNA and TRIM16 siRNA transfected cells treated with (B) MG132 (20 µM, 2h), where IF analysis was conducted with Ub and p62 antibodies. (C The graph shows the the percentage of cells with Ub-p62 co-localized dots. Data from ≥10 fields (40X), n=3, Mean ±SD, **p < 0.0003, *p < 0.002 (Student's unpaired t-test). Data information: Unless otherwise stated, scale bar: 10 µm.


D ) Representative confocal of HeLa TRIM16KO treated with ( D ) H2O2 ( 200 µM , 2h ) samples were processed for IF with Ub p62 antibody . E ) Representative confocal of control siRNA TRIM16 siRNA transfected treated with ( E ) H2O2 , ( 200 µM , 2h ) , where IF was conducted with Ub p62 antibodies . F ) graph shows of with Ub - p62 co - localized dots . Data from ≥ 10 fields ( 40X ) , n = 3 , Mean ± SD , * * p < 0 . 0003 , * p < 0 . 002 ( Student ' s unpaired t - ) . Data information : Unless otherwise stated , scale bar : 10 µm .

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-MEASURED_VAR 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 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 O O O O O O O O O O O O O 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) Representative confocal images of HeLa and TRIM16KO cells treated with (D) H2O2 (200 µM, 2h) and the samples were processed for IF analysis with Ub and p62 antibody. E) Representative confocal images of control siRNA and TRIM16 siRNA transfected cells treated with (E) H2O2, (200 µM, 2h), where IF analysis was conducted with Ub and p62 antibodies. F) The graph shows the the percentage of cells with Ub-p62 co-localized dots. Data from ≥10 fields (40X), n=3, Mean ±SD, **p < 0.0003, *p < 0.002 (Student's unpaired t-test). Data information: Unless otherwise stated, scale bar: 10 µm.


( G ) Western blot ( WB ) of detergent - soluble - insoluble fractions of HeLa TRIM16KO treated with MG132 ( 50 µM , 1h ) probed with indicated antibodies .

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

(G) Western blot (WB) analysis of detergent-soluble and -insoluble fractions of HeLa and TRIM16KO cells treated with MG132 (50 µM, 1h) and probed with indicated antibodies.


( H , I ) Densitometric ( using ImageJ ) of WB normalized with α - Tubulin or Lamin - B1 . Data information : Unless otherwise stated , 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

(H, I) Densitometric analysis (using ImageJ) of WB normalized with α-Tubulin or Lamin-B1. Data information: Unless otherwise stated, scale bar: 10 µm.


( J ) Representative confocal of HeLa TRIM16KO treated with MG132 ( 20 µM , 2h ) processed for IF with Ub LC3B antibody .

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-MEASURED_VAR O O

(J) Representative confocal images of HeLa and TRIM16KO cells treated with MG132 (20 µM, 2h) and processed for IF analysis with Ub and LC3B antibody.


( K , L ) WB of LC3B in lysates of HeLa TRIM16KO treated with ( K ) Bafilomycin A1 ( 300 nM , 3h ) alone or ( L ) with MG132 ( 10 µM , 4h ) as indicated . L . E , Low exposure ; H . E , high exposure .

O O O O O O O B-MEASURED_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

(K, L) WB analysis of LC3B levels in lysates of HeLa and TRIM16KO cells treated with (K) Bafilomycin A1 (300 nM, 3h) alone or (L) with MG132 (10 µM, 4h) as indicated. L.E, Low exposure; H.E, high exposure.


( M ) Representative confocal HeLa TRIM16KO treated with H2O2 ( 200 µM , 2h ) processed for IF with Proteostat dye , Ub , p62 antibody . To show details , brightness of TRIM16KO was increased more than control

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

(M) Representative confocal images HeLa and TRIM16KO cells treated with H2O2 (200 µM, 2h) and processed for IF analysis with Proteostat dye, Ub, and p62 antibody. To show the cellular details, the brightness of TRIM16KO cells images was increased more than control cells


( A ) Western blot of HeLa TRIM16KO lysates probed with indicated antibodies .

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

(A) Western blot analysis of HeLa and TRIM16KO cell lysates probed with indicated antibodies.


( B ) Western blot of lysates from HeLa TRIM16KO treated with or without MG132 probed with indicated antibodies .

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

(B) Western blot analysis of lysates from HeLa and TRIM16KO cells treated with or without MG132 and probed with indicated antibodies.


( C ) Left panel , western blotting with HeLa TRIM16KO lysates probed with indicated antibodies . Right panel , densitometric ( mean ± SD ) of band relative to actin , n = 3 , * * * p < 0 . 0005 ( Student ' s unpaired t - ) .

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

(C) Left panel, western blotting with HeLa and TRIM16KO cell lysates probed with indicated antibodies. Right panel, densitometric analysis (mean ±SD) of protein band intensity relative to actin, n=3, ***p < 0.0005 (Student's unpaired t-test).


D - G ( D , F ) HEK293T were either transiently transfected with an empty vector or a Flag - tagged - TRIM16 expressing vector immunoblotted with Flag or indicated antibodies . L . E , Low exposure ; H . E , High Exposure . ( E , G ) Densitometric ( mean ± SD ) of band relative to actin , n = 3 , * p < 0 . 05 ( Student ' s unpaired t - ) .

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

D-G (D, F) HEK293T cells were either transiently transfected with an empty vector or a Flag-tagged-TRIM16 expressing vector and immunoblotted with Flag or indicated antibodies. L.E, Low exposure; H.E, High Exposure. (E, G) Densitometric analysis (mean ±SD) of protein band intensity relative to actin, n=3, *p < 0.05 (Student's unpaired t-test).


( H ) WB of HeLa TRIM16KO lysates of treated with cycloheximide ( 100 µM ) for indicated period probed with different antibodies as indicated . blots of control TRIM16KO are exposed for equal duration developed together on same X - ray film . ( I ) of NRF2 , p62 , KEAP1 band intensities relative to actin . n = 3 , * * p < 0 . 005 , * p < 0 . 05 ( Student ' s unpaired t - ) .

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 B-CONTROLLED_VAR O 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 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

(H) WB analysis of HeLa and TRIM16KO lysates of cells treated with cycloheximide (100 µM) for the indicated period and probed with different antibodies as indicated. The blots of control and TRIM16KO cells are exposed for the equal duration and developed together on the same X-ray film. (I) Quantification of NRF2, p62, and KEAP1 band intensities relative to actin. n=3, **p < 0.005, *p < 0.05 (Student's unpaired t-test).


( J ) Immunoprecipitation ( IP ) of interaction between endogenous TRIM16 endogenous p62 in HeLa lysates in absence presence of MG132 ( 10 µM , 4h ) .

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 O

(J) Immunoprecipitation (IP) analysis of interaction between endogenous TRIM16 and endogenous p62 in HeLa cell lysates in absence and presence of MG132 (10 µM, 4h).


IP to map interaction between p62 TRIM16 domains in HEK293T lysates in ( L ) absence of MG132 ( 10 µM , 4h ) .

O O O O 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

IP analysis to map the interaction between p62 and TRIM16 domains in HEK293T cell lysates in (L) absence of MG132 (10 µM, 4h).


IP to map interaction between p62 TRIM16 domains in HEK293T lysates in ( M ) presence of MG132 ( 10 µM , 4h ) .

IP analysis to map the interaction between p62 and TRIM16 domains in HEK293T cell lysates in (M) presence of MG132 (10 µM, 4h).


( N ) IP of interaction between endogenous TRIM16 endogenous NRF2 in HeLa lysates . Mr , Molecular Marker .

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

(N) IP analysis of the interaction between endogenous TRIM16 and endogenous NRF2 in HeLa cell lysates. Mr, Molecular Weight Marker.


( O ) Co - IP to map interaction between NRF2 TRIM16 domains in HEK293T lysates .

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

(O) Co-IP analysis to map the interaction between NRF2 and TRIM16 domains in HEK293T cell lysates.


( P ) IP of interaction between endogenous TRIM16 endogenous KEAP1 in HeLa lysates .

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

(P) IP analysis of the interaction between endogenous TRIM16 and endogenous KEAP1 in HeLa cell lysates.


( A , B ) Co - IP of interaction between NRF2 KEAP1 in absence presence of TRIM16 .

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

(A, B) Co-IP analysis of the interaction between NRF2 and KEAP1 in absence and presence of TRIM16.


( C ) Co - IP of interaction between NRF2 p62 in absence presence of TRIM16 .

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

(C) Co-IP analysis of the interaction between NRF2 and p62 in absence and presence of TRIM16.


( D ) Co - IP of interaction between KEAP1 p62 in absence presence of TRIM16 .

(D) Co-IP analysis of the interaction between KEAP1 and p62 in absence and presence of TRIM16.


( A - C ) of NRF2 ubiquitination in absence presence of TRIM16 by Co - IP assays using transiently transfected plasmid constructs as indicated . Two different variants of ubiquitin are used , one that can only be ubiquitinated at Lysine 48 residue ( HA - K48 - UB ) other that can be only ubiquitinated at Lysine 63 residue ( HA - K63 - UB ) . other lysine residues are mutated . L . E , Low exposure ; H . E , High Exposure .

O O O O O O B-MEASURED_VAR 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

(A-C) Analysis of NRF2 ubiquitination in absence and presence of TRIM16 by Co-IP assays using transiently transfected plasmid constructs as indicated. Two different variants of ubiquitin protein are used, one that can only be ubiquitinated at Lysine 48 residue (HA-K48-UB) and other that can be only ubiquitinated at Lysine 63 residue (HA-K63-UB). All other lysine residues are mutated. L.E, Low exposure; H.E, High Exposure.


of NRF2 ubiquitination in absence presence of TRIM16 by Ni - NTA pull - down assays using transiently transfected plasmid constructs as indicated . His - tagged ubiquitin which is mutated at lysines except 63 position is used in these assays .

O B-MEASURED_VAR 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

Analysis of NRF2 ubiquitination in absence and presence of TRIM16 by Ni-NTA pull-down assays using transiently transfected plasmid constructs as indicated. His-tagged ubiquitin which is mutated at all lysines except 63 position is used in these assays.


( F ) of NRF2 ubiquitination in absence presence of TRIM16 deletion variants by Co - IP assays using transiently transfected plasmid constructs as indicated .

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

(F) Analysis of NRF2 ubiquitination in absence and presence of TRIM16 deletion variants by Co-IP assays using transiently transfected plasmid constructs as indicated.


( G ) Western blot of lysates from HeLa , TRIM16KO TRIM16KO complemented with TRIM16 deletion constructs where were treated with MG132 ( 20 µM , 2h ) blot is probed with indicated antibodies .

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

(G) Western blot analysis of lysates from HeLa, TRIM16KO cells and TRIM16KO cells complemented with TRIM16 deletion constructs where cells were treated with MG132 (20 µM, 2h) and the blot is probed with indicated antibodies.


( H ) Western blot of lysates from HeLa , TRIM16KO TRIM16KO complemented with TRIM16 deletion constructs where were treated with MG132 ( 20 µM , 2h ) blot is probed with indicated antibodies .

(H) Western blot analysis of lysates from HeLa, TRIM16KO cells and TRIM16KO cells complemented with TRIM16 deletion constructs where cells were treated with MG132 (20 µM, 2h) and the blot is probed with indicated antibodies.


( A ) Western blot of HeLa TRIM16KO lysates of treated with increasing concentrations of H2O2 as specified probed with antibodies as indicated .

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

(A) Western blot analysis of HeLa and TRIM16KO lysates of cells treated with increasing concentrations of H2O2 as specified and probed with antibodies as indicated.


( B ) WB of HeLa TRIM16KO lysates of treated with 400 µM of H2O2 for different durations as indicated probed with antibodies as shown . red arrows indicate time point where were retreated with 400 µM of H2O2 . L . E , Low exposure ; H . E , High Exposure .

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

(B) WB analysis of HeLa and TRIM16KO lysates of cells treated with 400 µM of H2O2 for different durations as indicated and probed with antibodies as shown. The red arrows indicate the time point where the cells were retreated with 400 µM of H2O2. L.E, Low exposure; H.E, High Exposure.


( C ) Densitometric of NRF2 band normalized with actin .

(C) Densitometric analysis of NRF2 band intensity normalized with actin.


( D ) WB of HeLa TRIM16KO lysates of treated with 2 . 5 µM ( 2 h ) of As2O3 .

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

(D) WB analysis of HeLa and TRIM16KO lysates of cells treated with 2.5 µM (2 h) of As2O3.


( E - G ) isolated from HeLa TRIM16KO , untreated or treated with MG132 ( 20 µM , 2h ) were subjected to qRT - PCR with primers of genes as indicated . fold induction in MG132 treated samples is calculated relative to untreated samples . Mean ± SD , n = 3 , * p < 0 . 05 , * * p < 0 . 005 ( Student ' s unpaired t - ) .

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

(E-G) RNA isolated from HeLa and TRIM16KO cells, untreated or treated with MG132 (20 µM, 2h) were subjected to qRT-PCR with primers of genes as indicated. The fold induction in MG132 treated samples is calculated relative to untreated samples. Mean ±SD, n=3, *p < 0.05, **p < 0.005 (Student's unpaired t-test).


isolated from control NRF2 knockdown HeLa transfected with control vector or Myc - TRIM16 subjected to qRT - PCR with primers of genes as indicated . Mean ± SD , n = 3 , * * p < 0 . 005 , * * * p < 0 . 0005 ( Student ' s unpaired t - ) .

O O O B-CONTROLLED_VAR 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

RNA isolated from control and NRF2 knockdown HeLa cells transfected with control vector or Myc-TRIM16 subjected to qRT-PCR with primers of genes as indicated. Mean ±SD, n=3, **p < 0.005, ***p < 0.0005 (Student's unpaired t-test).


( K ) Western blot of HeLa TRIM16KO lysates of treated with h increasing concentrations of H2O2 as specified probed with antibodies as indicated .

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

(K) Western blot analysis of HeLa and TRIM16KO lysates of cells treated with h increasing concentrations of H2O2 as specified and probed with antibodies as indicated.


( L ) Densitometric of Ub smear normalized with actin . Mean ± SD , n = 3 , * p < 0 . 05 ( Student ' s unpaired t - ) .

O O O O O O O O O O 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) Densitometric analysis of Ub smear intensity normalized with actin. Mean ±SD, n=3, *p < 0.05 (Student's unpaired t-test).


( M ) Representative confocal of control , Ubb Ube2n siRNA transfected treated with H2O2 ( 200 µM , 2h ) IF was performed with Ub p62 antibodies . Scale bar : 10 µm .

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

(M) Representative confocal images of control, Ubb and Ube2n siRNA transfected cells treated with H2O2 (200 µM, 2h) and IF was performed with Ub and p62 antibodies. Scale bar: 10 µm.


( N ) graph shows of with . Data from ≥ 10 microscopic fields for each condition ( 40X ) , n = 2 , Mean ± SD .

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

(N) The graph shows the percentage of cells with protein aggregates. Data from ≥ 10 microscopic fields for each condition (40X), n=2, Mean ±SD.


( O ) Western blot of Ubb Ube2n siRNA transfected lysates with antibodies as indicated .

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

(O) Western blot analysis of Ubb and Ube2n siRNA transfected cell lysates with antibodies as indicated.


( A ) Control or NRF2 siRNA transfected HeLa lysates were subjected to western blotting with indicated antibodies .

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

(A) Control or NRF2 siRNA transfected HeLa cells lysates were subjected to western blotting with indicated antibodies.


( B , C ) isolated from untreated or MG132 - treated ( 20 µM , 2h ) control or NRF2 siRNA transfected were subjected to qRT - PCR with primers of genes as indicated . fold induction in MG132 treated samples is calculated relative to untreated samples . Mean ± SD , n = 3 , * p < 0 . 05 ( Student ' s unpaired t - ) . Data information : Unless otherwise stated , scale bar : 10 µm .

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

(B, C) RNA isolated from untreated or MG132-treated (20 µM, 2h) control or NRF2 siRNA transfected cells were subjected to qRT-PCR with primers of genes as indicated. The fold induction in MG132 treated samples is calculated relative to untreated samples. Mean ±SD, n=3, *p < 0.05 (Student's unpaired t-test). Data information: Unless otherwise stated, scale bar: 10 µm.


WB of control siRNA or NRF2 siRNA transfected lysates of HeLa , untreated or treated with MG132 ( 20 µM , 2h ) as indicated probed with antibodies as shown . L . E , Low exposure ; H . E , High Exposure .

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

WB analysis of control siRNA or NRF2 siRNA transfected lysates of HeLa cells, untreated or treated with MG132 (20 µM, 2h) as indicated and probed with antibodies as shown. L.E, Low exposure; H.E, High Exposure.


WB of control siRNA or NRF2 siRNA transfected lysates of HeLa , untreated or treated with H2O2 ( 200 µM , 2h ) as indicated probed with antibodies as shown . L . E , Low exposure ; H . E , High Exposure .

WB analysis of control siRNA or NRF2 siRNA transfected lysates of HeLa cells, untreated or treated with H2O2 (200 µM, 2h) as indicated and probed with antibodies as shown. L.E, Low exposure; H.E, High Exposure.


Left panels : Representative confocal of control siRNA NRF2 siRNA transfected treated with ( F ) MG132 ( 20 µM , 2h ) , where IF was conducted with Ub p62 antibodies . Right panels : tracing corresponding to a in zoom panel .

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-MEASURED_VAR O O O O O O O O O O O O O

Left panels: Representative confocal images of control siRNA and NRF2 siRNA transfected cells treated with (F) MG132 (20 µM, 2h), where IF was conducted with Ub and p62 antibodies. Right panels: fluorescence intensity line tracing corresponding to a white line in zoom panel.


Left panels : Representative confocal of control siRNA NRF2 siRNA transfected treated with ( G ) H2O2 ( 200 µM , 2h ) , where IF was conducted with Ub p62 antibodies . Right panels : tracing corresponding to a in zoom panel .

Left panels: Representative confocal images of control siRNA and NRF2 siRNA transfected cells treated with (G) H2O2 (200 µM, 2h), where IF was conducted with Ub and p62 antibodies. Right panels: fluorescence intensity line tracing corresponding to a white line in zoom panel.


( H ) Hela , TRIM16KO , TRIM16KO complemented with Myc - NRF2 or GFP - NRF2 were treated with MG132 ( 20 µM , 2h ) subjected to WB with Myc , GFP indicated antibodies .

O O O O O B-CONTROLLED_VAR 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

(H) Hela cells, TRIM16KO cells, and TRIM16KO cells complemented with Myc-NRF2 or GFP-NRF2 were treated with MG132 (20 µM, 2h) and subjected to WB analysis with Myc, GFP and indicated antibodies.


( I ) Representative confocal of Hela , TRIM16KO , TRIM16KO complemented with Myc - NRF2 which were treated with MG132 ( 20 µM , 2h ) subjected to IF with Ub p62 antibodies .

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 B-MEASURED_VAR O O

(I) Representative confocal images of Hela cells, TRIM16KO cells, and TRIM16KO cells complemented with Myc-NRF2 which were treated with MG132 (20 µM, 2h) and subjected to IF with Ub and p62 antibodies.


( J ) Graph represent % of with Ub - p62 dots , data from ≥ 10 microscopic fields ( 40X ) , n = 3 , mean ± SD , * p < 0 . 005 ( one - way ANOVA ) . Data information : Unless otherwise stated , scale bar : 10 µm .

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

(J) Graph represent % of cells with Ub-p62 dots, data from ≥ 10 microscopic fields (40X), n=3, mean ±SD, *p < 0.005 (one-way ANOVA test). Data information: Unless otherwise stated, scale bar: 10 µm.


WB of detergent - soluble - insoluble fractions of HeLa TRIM16KO expressing either ( A ) Flag - p62 probed with Flag or indicated antibodies . L . E , Low exposure ; H . E , High Exposure . Densitometric of WB ' s normalized with α - Tubulin or Lamin - B1 .

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

WB analysis of detergent-soluble and -insoluble fractions of HeLa and TRIM16KO cells expressing either (A) Flag-p62 probed with Flag or indicated antibodies. L.E, Low exposure; H.E, High Exposure. Densitometric analysis of WB's normalized with α-Tubulin or Lamin-B1.


WB of detergent - soluble - insoluble fractions of HeLa TRIM16KO expressing either ( C ) GFP - polyQ74 probed with or GFP or indicated antibodies . L . E , Low exposure ; H . E , High Exposure . Densitometric of WB ' s normalized with α - Tubulin or Lamin - B1 .

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

WB analysis of detergent-soluble and -insoluble fractions of HeLa and TRIM16KO cells expressing either (C) GFP-polyQ74 probed with or GFP or indicated antibodies. L.E, Low exposure; H.E, High Exposure. Densitometric analysis of WB's normalized with α-Tubulin or Lamin-B1.


( E ) Confocal of GFP - TRIM16 expressing HeLa , untreated or treated with MG132 ( 10 µM , 4h ) / or Bafilomycin A1 ( 300 nM , 3h ) IF is performed with antibodies as indicated . Right panel graphs : tracing corresponding to a in zoom panel . Scale bar : 10 µm . ( F ) graph shows of with TRIM16 - Ub or TRIM16 - Ub - p62 co - localized dots / . Data from ≥ 10 microscopic fields , n = 2 , mean ± SD .

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 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

(E) Confocal images of GFP-TRIM16 expressing HeLa cells, untreated or treated with MG132 (10 µM, 4h) and/or Bafilomycin A1 (300 nM, 3h) and IF analysis is performed with antibodies as indicated. Right panel graphs: fluorescence intensity line tracing corresponding to a white line in zoom panel. Scale bar: 10 µm. (F) The graph shows the percentage of cells with TRIM16-Ub or TRIM16-Ub-p62 co-localized dots/aggregates. Data from ≥ 10 microscopic fields, n=2, mean ±SD.


( G ) Representative confocal of GFP - TRIM16 expressing HeLa , untreated or treated with MG132 ( 10 µM , 4h ) / or Bafilomycin A1 ( 300 nM , 3h ) IF is performed with antibodies as indicated . Right panel graphs : tracing corresponding to a in zoom panel . Scale bar : 10 µm .

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

(G) Representative confocal images of GFP-TRIM16 expressing HeLa cells, untreated or treated with MG132 (10 µM, 4h) and/or Bafilomycin A1 (300 nM, 3h) and IF analysis is performed with antibodies as indicated. Right panel graphs: fluorescence intensity line tracing corresponding to a white line in zoom panel. Scale bar: 10 µm.


( H ) Co - IP of interaction between two differently tagged TRIM16 variants .

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

(H) Co-IP analysis of the interaction between two differently tagged TRIM16 variants.


( I ) WB of detergent - soluble - insoluble DSS - cross - linked fractions of HeLa TRIM16KO , untreated or treated with MG132 ( 10 µM , 4h ) probed with indicated antibodies .

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

(I) WB analysis of detergent-soluble and -insoluble DSS-cross-linked fractions of HeLa and TRIM16KO cells, untreated or treated with MG132 (10 µM, 4h) and probed with indicated antibodies.


( J ) Co - IP of interaction between Flag - tagged TRIM16 Myc - tagged TRIM16 full its domain deletion .

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

(J) Co-IP analysis of the interaction between Flag-tagged TRIM16 and Myc-tagged TRIM16 full length and its domain deletion proteins.


( A ) MTT assays performed at different time points with HeLa TRIM16KO , untreated or treated with H2O2 ( 400 µM , 2h ) . Data , mean ± SD , n = 3 , * p < 0 . 05 ( Student ' s unpaired t - ) .

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

(A) MTT assays performed at different time points with HeLa and TRIM16KO cells, untreated or treated with H2O2 (400 µM, 2h). Data, mean ±SD, n=3, *p < 0.05 (Student's unpaired t-test).


( B ) of HeLa TRIM16KO , untreated or treated with As2O3 ( 2 . 5 µM , 4h ) . Scale bar : 400 µm .

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) Images of HeLa and TRIM16KO cells, untreated or treated with As2O3 (2.5 µM, 4h). Scale bar: 400 µm.


Flow cytometry of HeLa TRIM16KO stained with Annexin - V / Propidium Iodide ( double ) , untreated or treated with ( C ) As2O3 ( 2 . 5 µM , 4h )

O O O O B-CONTROLLED_VAR 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

Flow cytometry analysis of HeLa and TRIM16KO cells stained with Annexin-V/Propidium Iodide (double staining), untreated or treated with (C) As2O3 (2.5 µM, 4h)


Flow cytometry of HeLa TRIM16KO stained with Annexin - V / Propidium Iodide ( double ) , untreated or treated with ( D ) H2O2 ( 400 µM , 2h )

O O O O B-CONTROLLED_VAR 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

Flow cytometry analysis of HeLa and TRIM16KO cells stained with Annexin-V/Propidium Iodide (double staining), untreated or treated with (D) H2O2 (400 µM, 2h)


Flow cytometry of HeLa TRIM16KO stained with Annexin - V / Propidium Iodide ( double ) , untreated or treated with ( E ) puromycin ( 5 µg / ml , 6h )

Flow cytometry analysis of HeLa and TRIM16KO cells stained with Annexin-V/Propidium Iodide (double staining), untreated or treated with (E) puromycin (5 µg/ml, 6h)


Flow cytometry of HeLa TRIM16KO stained with Annexin - V / Propidium Iodide ( double ) , untreated or treated with ( F ) MG132 ( 20 µM , 8h ) .

O O O O B-CONTROLLED_VAR 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

Flow cytometry analysis of HeLa and TRIM16KO cells stained with Annexin-V/Propidium Iodide (double staining), untreated or treated with (F) MG132 (20 µM, 8h).


( G ) Immunoblot blot of HeLa TRIM16KO lysates of treated with 5 µM of As2O3 for different durations as indicated probed with antibodies as shown . L . E , Low exposure ; H . E , High Exposure . Arrowheads indicate form of PARP - 1 Casapse - 3 .

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

(G) Immunoblot blot analysis of HeLa and TRIM16KO lysates of cells treated with 5 µM of As2O3 for different durations as indicated and probed with antibodies as shown. L.E, Low exposure; H.E, High Exposure. Arrowheads indicate the cleaved form of PARP-1 and Casapse-3.


( A , B ) Clonogenic assays were performed with HeLa TRIM16KO , untreated or treated with As2O3 or MG132 .

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

(A, B) Clonogenic assays were performed with HeLa and TRIM16KO cells, untreated or treated with As2O3 or MG132.


( C ) volumes of HeLa TRIM16KO tumors at indicated time points . As2O3 is injected when volume of groups was > 100mm3 ( day 0 in graph ) . volumes were measured on an interval as indicated . Mean ± SE , n = 6 ( each group ) , * p < 0 . 05 , * * p < 0 . 005 , * * * p < 0 . 0005 ( ANOVA ) . ( D ) Graph shows % regression ( volume on day 1 of As2O3 treatment / volume on day of sacrifice X 100 ) . Mean ± SE , n = 6 ( each group ) , * * p < 0 . 005 ( Student ' s unpaired t - ) .

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

(C) Tumor volumes of HeLa and TRIM16KO tumors at indicated time points. As2O3 is injected when tumor volume of all groups was >100mm3 (day 0 in the graph). Tumor volumes were measured on an interval as indicated. Mean ±SE, n=6 (each group), *p < 0.05, **p < 0.005, ***p < 0.0005 (ANOVA). (D) Graph shows the % tumor regression (Tumor volume on day 1 of As2O3 treatment / Tumor volume on day of sacrifice X 100). Mean ±SE, n=6 (each group), **p < 0.005 (Student's unpaired t-test).


( E ) Representative of HeLa TRIM16KO tumors formed in nude mice in absence presence of As2O3 . ( F ) Representative pictures of dissected tumors .

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

(E) Representative images of HeLa and TRIM16KO tumors formed in the nude mice in the absence and presence of As2O3. (F) Representative pictures of dissected tumors.


( G ) Immunohistochemistry performed with KI - 67 proliferation marker hematoxylin stain . graph represents % of with KI - 67 . > 400 were counted for this from three different sections from different , mean ± SD , * p < 0 . 05 ( Student ' s unpaired t - ) . Scale bar : 50 µm .

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

(G) Immunohistochemistry analysis performed with KI-67 cell proliferation marker and hematoxylin nuclear stain. The graph represents the % of cells with KI-67 staining. >400 cells were counted for this analysis from three different sections from different animals, mean ±SD, *p < 0.05 (Student's unpaired t-test). Scale bar: 50 µm.


( H ) WB of tissue lysates from two different mice with antibodies as indicated . L . E , Low exposure ; H . E , High Exposure .

O O O O O O 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) WB analysis of tumor tissue lysates from two different mice with antibodies as indicated. L.E, Low exposure; H.E, High Exposure.


( A ) Western blotting for phospho - Thr172 - AMPK ( pAMPK ) , total AMPK , phospho - Ser79 - ACC ( pACC ) , total ACC 24h after treatment . represents pAMPK / AMPK pACC / ACC ratios relative to non - treated ( NT ) control Data information : quantifications are of three independent experiments ( n = 3 ) error bars represent standard error of mean ( ) . Significance between means was first determined using ANOVA . Significance p - values were calculated using Fisher " s LSD . * , p < 0 . 05 ; * * , p < 0 . 01 from NT controls ; † † , p < 0 . 01 from IFNγ / TNFα treated controls

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

(A) Western blotting for phospho-Thr172-AMPK (pAMPK), total AMPK, phospho-Ser79-ACC (pACC), and total ACC 24h after treatment. Quantification represents the pAMPK/AMPK and pACC/ACC ratios relative to the non-treated (NT) control Data information: All quantifications are of three independent experiments (n = 3) and error bars represent standard error of the mean (SEM). Significance between means was first determined using ANOVA. Significance p-values were calculated using Fisher"s LSD. *, p < 0.05; **, p < 0.01 from NT controls; ††, p < 0.01 from IFNγ/TNFα treated controls


( B ) Phase contrast of 72h after treatment . Scale bars represent 0 . 25mm

(B) Phase contrast images of fibers 72h after treatment. Scale bars represent 0.25mm


( C ) Immunofluorescence for myoglobin myosin heavy chain ( MyHC ) 48h after treatment . Scale bars represent 50µm . represents average myotube width Data information : quantifications are of three independent experiments ( n = 3 ) error bars represent standard error of mean ( ) . Significance between means was first determined using ANOVA . Significance p - values were calculated using Fisher " s LSD . * , p < 0 . 05 ; * * , p < 0 . 01 from NT controls ; † † , p < 0 . 01 from IFNγ / TNFα treated controls

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

(C) Immunofluorescence staining for myoglobin and myosin heavy chain (MyHC) 48h after treatment. Scale bars represent 50µm. Quantification represents the average myotube width Data information: All quantifications are of three independent experiments (n = 3) and error bars represent standard error of the mean (SEM). Significance between means was first determined using ANOVA. Significance p-values were calculated using Fisher"s LSD. *, p < 0.05; **, p < 0.01 from NT controls; ††, p < 0.01 from IFNγ/TNFα treated controls


( D - E ) RT - qPCR of mRNA of MyoD ( D 24h after treatment relative to NT control . Data information : quantifications are of three independent experiments ( n = 3 ) error bars represent standard error of mean ( ) . Significance between means was first determined using ANOVA . Significance p - values were calculated using Fisher " s LSD . * , p < 0 . 05 ; * * , p < 0 . 01 from NT controls ; † † , p < 0 . 01 from IFNγ / TNFα treated controls

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 O O O 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

(D-E) RT-qPCR analysis of the mRNA levels of MyoD (D 24h after treatment relative to the NT control. Data information: All quantifications are of three independent experiments (n = 3) and error bars represent standard error of the mean (SEM). Significance between means was first determined using ANOVA. Significance p-values were calculated using Fisher"s LSD. *, p < 0.05; **, p < 0.01 from NT controls; ††, p < 0.01 from IFNγ/TNFα treated controls


( D - E ) RT - qPCR of mRNA o myogenin ( E ) 24h after treatment relative to NT control . Data information : quantifications are of three independent experiments ( n = 3 ) error bars represent standard error of mean ( ) . Significance between means was first determined using ANOVA . Significance p - values were calculated using Fisher " s LSD . * , p < 0 . 05 ; * * , p < 0 . 01 from NT controls ; † † , p < 0 . 01 from IFNγ / TNFα treated controls

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 O O O O 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

(D-E) RT-qPCR analysis of the mRNA levels o myogenin (E) 24h after treatment relative to the NT control. Data information: All quantifications are of three independent experiments (n = 3) and error bars represent standard error of the mean (SEM). Significance between means was first determined using ANOVA. Significance p-values were calculated using Fisher"s LSD. *, p < 0.05; **, p < 0.01 from NT controls; ††, p < 0.01 from IFNγ/TNFα treated controls


( A - B ) Rates of glucose consumption ( A measured in media 24h after treatment relative to non - treated ( NT ) control from three independent experiments ( n = 3 ) . Error bars represent Data information : Significance between means was first determined using ANOVA . Significance p - values were calculated using Fisher " s LSD . * , p < 0 . 05 ; * * , p < 0 . 01 from NT controls

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Rates of glucose consumption (A measured in the media 24h after treatment relative to the non-treated (NT) control from three independent experiments (n = 3). Error bars represent the SEM Data information: Significance between means was first determined using ANOVA. Significance p-values were calculated using Fisher"s LSD. *, p < 0.05; **, p < 0.01 from NT controls


( A ) Western blotting of phospho - Thr389 - p70S6K ( pS6K ) , total p70S6K ( S6K ) , phospho - Ser235 / 236 - S6 ( pS6 ) total S6 . represents pS6K / S6K ( n = 3 ) pS6 / S6 ( n = 4 ) ratios relative to non - treated ( NT ) control Data information : Error bars represent . Significance between means was first determined using ANOVA . Significance p - values were calculated using Fisher " s LSD . * , p < 0 . 05 ; * * , p < 0 . 01 from NT controls ; † † , p < 0 . 01 from IFNγ / TNFα treated

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

(A) Western blotting of phospho-Thr389-p70S6K (pS6K), total p70S6K (S6K), phospho-Ser235/236-S6 (pS6) and total S6. Quantification represents the pS6K/S6K (n = 3) and pS6/S6 (n = 4) ratios relative to the non-treated (NT) control Data information: Error bars represent the SEM. Significance between means was first determined using ANOVA. Significance p-values were calculated using Fisher"s LSD. *, p < 0.05; **, p < 0.01 from NT controls; ††, p < 0.01 from IFNγ/TNFα treated controls


( B ) Radiographic of de novo synthesis using 35S - labelling . represents whole lane radiation signal density standardized to Coomassie relative to NT control ( n = 3 ) . Data information : Error bars represent . Significance between means was first determined using ANOVA . Significance p - values were calculated using Fisher " s LSD . * , p < 0 . 05 ; * * , p < 0 . 01 from NT controls ; † † , p < 0 . 01 from IFNγ / TNFα treated controls

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

(B) Radiographic analysis of de novo protein synthesis using 35S-labelling. Quantification represents whole lane radiation signal density standardized to Coomassie staining and relative to NT control levels (n = 3). Data information: Error bars represent the SEM. Significance between means was first determined using ANOVA. Significance p-values were calculated using Fisher"s LSD. *, p < 0.05; **, p < 0.01 from NT controls; ††, p < 0.01 from IFNγ/TNFα treated controls


( A ) Immunofluorescence for myoglobin myosin heavy chain ( MyHC ) 48h after treatment . Scale bars represent 50µm . represents average myotube width Data information : quantifications are of three independent experiments ( n = 3 ) error bars represent . For panels A through D , significance between means was first determined using ANOVA . Significance p - values were calculated using Fisher " s LSD

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

(A) Immunofluorescence staining for myoglobin and myosin heavy chain (MyHC) 48h after treatment. Scale bars represent 50µm. Quantification represents the average myotube width Data information: All quantifications are of three independent experiments (n = 3) and error bars represent the SEM. For panels A through D, significance between means was first determined using ANOVA. Significance p-values were calculated using Fisher"s LSD


( B - C ) Rates of glucose consumption ( B measured in media 24h after treatment relative to DMSO treated control Data information : quantifications are of three independent experiments ( n = 3 ) error bars represent . For panels A through D , significance between means was first determined using ANOVA . Significance p - values were calculated using Fisher " s LSD

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-C) Rates of glucose consumption (B measured in the media 24h after treatment relative to the DMSO treated control Data information: All quantifications are of three independent experiments (n = 3) and error bars represent the SEM. For panels A through D, significance between means was first determined using ANOVA. Significance p-values were calculated using Fisher"s LSD


( B - C ) Rates o lactate production ( C ) measured in media 24h after treatment relative to DMSO treated control Data information : quantifications are of three independent experiments ( n = 3 ) error bars represent . For panels A through D , significance between means was first determined using ANOVA . Significance p - values were calculated using Fisher " s LSD

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-C) Rates o lactate production (C) measured in the media 24h after treatment relative to the DMSO treated control Data information: All quantifications are of three independent experiments (n = 3) and error bars represent the SEM. For panels A through D, significance between means was first determined using ANOVA. Significance p-values were calculated using Fisher"s LSD


( D ) Western blotting of phospho - Thr389 - p70S6K ( pS6K ) , total p70S6K ( S6K ) , phospho - Ser235 / 236 - S6 ( pS6 ) total S6 . represents pS6K / S6K pS6 / S6 ratios relative to DMSO treated control Data information : quantifications are of three independent experiments ( n = 3 ) error bars represent . For panels A through D , significance between means was first determined using ANOVA . Significance p - values were calculated using Fisher " s LSD

O O O O O O O O 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 O B-MEASURED_VAR O B-MEASURED_VAR O O B-MEASURED_VAR 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 O O O O O O O O O O O O O O O O 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) Western blotting of phospho-Thr389-p70S6K (pS6K), total p70S6K (S6K), phospho-Ser235/236-S6 (pS6) and total S6. Quantification represents the pS6K/S6K and pS6/S6 ratios relative to the DMSO treated control Data information: All quantifications are of three independent experiments (n = 3) and error bars represent the SEM. For panels A through D, significance between means was first determined using ANOVA. Significance p-values were calculated using Fisher"s LSD


( E ) Western blotting of iNOS , tubulin , phospho - Thr172 AMPKα ( pAMPK ) , total AMPKα ( AMPK ) , phospho - Ser79 - ACC ( pACC ) total ACC . represents of iNOS normalized to tubulin relative to IFNγ / TNFα control Data information : quantifications are of three independent experiments ( n = 3 ) error bars represent For panels E through G , p - values were calculated using Student " s t - . * , p < 0 . 05 ; * * , p < 0 . 01 from DMSO controls ; † † , p < 0 . 01 from IFNγ / TNFα treated

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

(E) Western blotting of iNOS, tubulin, phospho-Thr172 AMPKα (pAMPK), total AMPKα (AMPK), phospho-Ser79-ACC (pACC) and total ACC. Quantification represents the levels of iNOS protein normalized to tubulin and relative to the IFNγ/TNFα control Data information: All quantifications are of three independent experiments (n = 3) and error bars represent the SEM For panels E through G, p-values were calculated using the Student"s t-test. *, p < 0.05; **, p < 0.01 from DMSO controls; ††, p < 0.01 from IFNγ/TNFα treated controls


( F ) RT - qPCR of iNOS mRNA relative to IFNγ / TNFα control 24h after treatment Data information : quantifications are of three independent experiments ( n = 3 ) error bars represent For panels E through G , p - values were calculated using Student " s t - . * , p < 0 . 05 ; * * , p < 0 . 01 from DMSO controls ; † † , p < 0 . 01 from IFNγ / TNFα treated

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

(F) RT-qPCR analysis of iNOS mRNA levels relative to the IFNγ/TNFα control 24h after treatment Data information: All quantifications are of three independent experiments (n = 3) and error bars represent the SEM For panels E through G, p-values were calculated using the Student"s t-test. *, p < 0.05; **, p < 0.01 from DMSO controls; ††, p < 0.01 from IFNγ/TNFα treated controls


( G ) Nitric oxide ( NO ) in culture media of relative to IFNγ / TNFα control 24h after treatment Data information : quantifications are of three independent experiments ( n = 3 ) error bars represent For panels E through G , p - values were calculated using Student " s t - . * , p < 0 . 05 ; * * , p < 0 . 01 from DMSO controls ; † † , p < 0 . 01 from IFNγ / TNFα treated

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

(G) Nitric oxide (NO) levels in the culture media of cells relative to the IFNγ/TNFα control 24h after treatment Data information: All quantifications are of three independent experiments (n = 3) and error bars represent the SEM For panels E through G, p-values were calculated using the Student"s t-test. *, p < 0.05; **, p < 0.01 from DMSO controls; ††, p < 0.01 from IFNγ/TNFα treated controls


( A ) Immunofluorescence for myoglobin myosin heavy chain ( MyHC ) 48h after treatment . Scale bars represent 50µm . represents average myotube width Data information : quantifications are of three independent experiments ( n = 3 ) error bars represent . Significance between means was first determined using ANOVA . Significance p - values were calculated using Fisher " s LSD . * , p < 0 . 05 ; * * , p < 0 . 01 from non - treated ( NT ) controls ; † , p < 0 . 05 ; † † , p < 0 . 01 from IFNγ / TNFα treated controls

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

(A) Immunofluorescence staining for myoglobin and myosin heavy chain (MyHC) 48h after treatment. Scale bars represent 50µm. Quantification represents the average myotube width Data information: All quantifications are of three independent experiments (n = 3) and error bars represent the SEM. Significance between means was first determined using ANOVA. Significance p-values were calculated using Fisher"s LSD. *, p < 0.05; **, p < 0.01 from non-treated (NT) controls; †, p < 0.05; ††, p < 0.01 from IFNγ/TNFα treated controls


( B ) Western blotting for iNOS tubulin 24h after treatment . represents of iNOS normalized to tubulin relative to IFNγ / TNFα control Data information : quantifications are of three independent experiments ( n = 3 ) error bars represent . Significance between means was first determined using ANOVA . Significance p - values were calculated using Fisher " s LSD . * , p < 0 . 05 ; * * , p < 0 . 01 from non - treated ( NT ) controls ; † , p < 0 . 05 ; † † , p < 0 . 01 from IFNγ / TNFα treated controls

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

(B) Western blotting for iNOS and tubulin protein levels 24h after treatment. Quantification represents the levels of iNOS protein normalized to tubulin and relative to the IFNγ/TNFα control Data information: All quantifications are of three independent experiments (n = 3) and error bars represent the SEM. Significance between means was first determined using ANOVA. Significance p-values were calculated using Fisher"s LSD. *, p < 0.05; **, p < 0.01 from non-treated (NT) controls; †, p < 0.05; ††, p < 0.01 from IFNγ/TNFα treated controls


( C ) Western blotting of phospho - Thr389 - p70S6K ( pS6K ) , total p70S6K ( S6K ) , phospho - Ser235 / 236 - S6 ( pS6 ) total S6 . represents pS6K / S6K pS6 / S6 ratios relative to NT control . Data information : quantifications are of three independent experiments ( n = 3 ) error bars represent . Significance between means was first determined using ANOVA . Significance p - values were calculated using Fisher " s LSD . * , p < 0 . 05 ; * * , p < 0 . 01 from non - treated ( NT ) controls ; † , p < 0 . 05 ; † † , p < 0 . 01 from IFNγ / TNFα treated controls

O O O O O O O O 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 O B-MEASURED_VAR O B-MEASURED_VAR O O B-MEASURED_VAR 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Western blotting of phospho-Thr389-p70S6K (pS6K), total p70S6K (S6K), phospho-Ser235/236-S6 (pS6) and total S6. Quantification represents the pS6K/S6K and pS6/S6 ratios relative to the NT control. Data information: All quantifications are of three independent experiments (n = 3) and error bars represent the SEM. Significance between means was first determined using ANOVA. Significance p-values were calculated using Fisher"s LSD. *, p < 0.05; **, p < 0.01 from non-treated (NT) controls; †, p < 0.05; ††, p < 0.01 from IFNγ/TNFα treated controls


( A ) Representative of overall hind limb musculature . Scale bar represents 1cm

(A) Representative images of overall hind limb musculature. Scale bar represents 1cm


( B ) Western blotting for phospho - Ser79 - ACC ( pACC ) total ACC

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

(B) Western blotting for phospho-Ser79-ACC (pACC) and total ACC


( C ) Cross - sectional of gastrocnemius muscle calculated from 250 per mouse from four mice per cohort ( n = 4 ) . Cross - sections were stained by H & E . Scale bars represent 100µm Data information : Error bars represent . Significance between means was first determined using ANOVA . Significance p - values were calculated using Fisher " s LSD . * , p < 0 . 05 ; * * , p < 0 . 01 from saline controls ; † , p < 0 . 05 ; † † , p < 0 . 01 from C26 controls

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Cross-sectional analysis of the gastrocnemius muscle calculated from 250 fibers per mouse from four mice per cohort (n = 4). Cross-sections were stained by H&E. Scale bars represent 100µm Data information: Error bars represent the SEM. Significance between means was first determined using ANOVA. Significance p-values were calculated using Fisher"s LSD. *, p < 0.05; **, p < 0.01 from saline controls; †, p < 0.05; ††, p < 0.01 from C26 controls


( D - E ) RT - qPCR of Atrogin - 1 / MAFbx ( D mRNA expression from tibialis anterior of three mice per cohort ( n = 3 ) . Data information : Error bars represent . Significance between means was first determined using ANOVA . Significance p - values were calculated using Fisher " s LSD . * , p < 0 . 05 ; * * , p < 0 . 01 from saline controls ; † , p < 0 . 05 ; † † , p < 0 . 01 from C26 controls

O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-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 O O O O O O O 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-E) RT-qPCR analysis of Atrogin-1/MAFbx (D mRNA expression from the tibialis anterior of three mice per cohort (n = 3). Data information: Error bars represent the SEM. Significance between means was first determined using ANOVA. Significance p-values were calculated using Fisher"s LSD. *, p < 0.05; **, p < 0.01 from saline controls; †, p < 0.05; ††, p < 0.01 from C26 controls


( D - E ) RT - qPCR o MuRF1 ( E ) mRNA expression from tibialis anterior of three mice per cohort ( n = 3 ) . Data information : Error bars represent . Significance between means was first determined using ANOVA . Significance p - values were calculated using Fisher " s LSD . * , p < 0 . 05 ; * * , p < 0 . 01 from saline controls ; † , p < 0 . 05 ; † † , p < 0 . 01 from C26 controls

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 O O O O O 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-E) RT-qPCR analysis o MuRF1 (E) mRNA expression from the tibialis anterior of three mice per cohort (n = 3). Data information: Error bars represent the SEM. Significance between means was first determined using ANOVA. Significance p-values were calculated using Fisher"s LSD. *, p < 0.05; **, p < 0.01 from saline controls; †, p < 0.05; ††, p < 0.01 from C26 controls


( A ) Cross - sectional of gastrocnemius muscle calculated from 250 per mouse from four mice per cohort ( n = 4 ) . Cross - sections were stained by H & E . Scale bars represent 100µm Data information : Error bars represent . Significance between means was first determined using ANOVA . Significance p - values were calculated using Fisher " s LSD . * , p < 0 . 05 ; * * , p < 0 . 01 from PF controls

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Cross-sectional analysis of the gastrocnemius muscle calculated from 250 fibers per mouse from four mice per cohort (n = 4). Cross-sections were stained by H&E. Scale bars represent 100µm Data information: Error bars represent the SEM. Significance between means was first determined using ANOVA. Significance p-values were calculated using Fisher"s LSD. *, p < 0.05; **, p < 0.01 from PF controls


( B - C ) RT - qPCR of Atrogin - 1 / MAFbx ( B mRNA expression from tibialis anterior of four mice per cohort ( n = 4 ) . Data information : Error bars represent . Significance between means was first determined using ANOVA . Significance p - values were calculated using Fisher " s LSD . * , p < 0 . 05 ; * * , p < 0 . 01 from PF controls

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

(B-C) RT-qPCR analysis of Atrogin-1/MAFbx (B mRNA expression from the tibialis anterior of four mice per cohort (n = 4). Data information: Error bars represent the SEM. Significance between means was first determined using ANOVA. Significance p-values were calculated using Fisher"s LSD. *, p < 0.05; **, p < 0.01 from PF controls


( B - C ) RT - qPCR o MuRF1 ( C ) mRNA expression from tibialis anterior of four mice per cohort ( n = 4 ) . Data information : Error bars represent . Significance between means was first determined using ANOVA . Significance p - values were calculated using Fisher " s LSD . * , p < 0 . 05 ; * * , p < 0 . 01 from PF controls

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 O O O O O O O O O

(B-C) RT-qPCR analysis o MuRF1 (C) mRNA expression from the tibialis anterior of four mice per cohort (n = 4). Data information: Error bars represent the SEM. Significance between means was first determined using ANOVA. Significance p-values were calculated using Fisher"s LSD. *, p < 0.05; **, p < 0.01 from PF controls


Representative grey - scale of rat hippocampal neurons ( DIV 10 ) transfected with GFP indicated miRNA mimics . of mean number of intersections by Sholl . GFP - only transfected conditions were set to one in each experiment .

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

Representative grey-scale images of primary rat hippocampal neurons (DIV 10) transfected with GFP and the indicated miRNA mimics. Quantification of the mean number of intersections by Sholl analysis. GFP-only transfected conditions were set to one in each experiment.


Representative grey - scale of GFP - transfected Cacna1c + / + ( WT ) or Cacna1c + / - rat hippocampal neurons ( DIV 10 ) . Scale bars = 50 μm . of mean number of intersections by Sholl . Data are represented as scattered dot plots with bar , mean ± S . D . ( n = 3 independent experiments ; Unpaired two - sample t - , * * p = 0 . 0096 ) . Fold changes represent changes in dendritic complexity relative to control condition .

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

Representative grey-scale images of GFP-transfected Cacna1c+/+ (WT) or Cacna1c+/- primary rat hippocampal neurons (DIV 10). Scale bars = 50 μm.   Quantification of the mean number of intersections by Sholl analysis. Data are represented as scattered dot plots with bar, mean±S.D. (n=3 independent experiments; Unpaired two-sample t-Test, **p=0.0096). Fold changes represent changes in dendritic complexity relative to the control condition.


Representative of DIV 10 hippocampal neurons co - transfected with control or miR - 499 - 5p mimics a pMT2 - CACNB2 expression plasmid ( CACNB2 ) . Scale bars = 50 μm . of mean number of intersections by Sholl . Data are represented as scattered dot plots with bar , mean ± S . D . ( n = 3 independent experiments ; Two - way ANOVA : no main effect of CACNB2 transfection p = 0 . 5027 or miRNA mimics p = 0 . 0805 , main effect of CACBN2 transfection by miRNA mimics interaction p = 0 . 0213 , Tukey ' s HSD : * p = 0 . 0303 ) . Fold changes represent changes in dendritic complexity of transfected neurons compared to non - treated control neurons . ns = not significant .

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

Representative images of DIV 10 hippocampal neurons co-transfected with control or miR-499-5p mimics and a pMT2-CACNB2 expression plasmid (CACNB2). Scale bars = 50 μm.   Quantification of the mean number of intersections by Sholl analysis. Data are represented as scattered dot plots with bar, mean±S.D. (n=3 independent experiments; Two-way ANOVA: no main effect of the CACNB2 transfection p=0.5027 or the miRNA mimics p=0.0805, main effect of the CACBN2 transfection by miRNA mimics interaction p=0.0213, Tukey's HSD: *p=0.0303). Fold changes represent the changes in dendritic complexity of transfected neurons compared to non-treated control neurons. ns = not significant.


A ) Representative of DIV 19 rat hippocampal neurons co - transfected with GFP ( green channel ) Cav1 . 2 - HA ( red channel ) , together with either control or miR - 499 - 5p mimics . After 12 - 13 days of expression , labeling with Anti - HA antibodies was performed in live conditions to identify surface Cav1 . 2 channels . Whole : scale bars = 20 μm ; dendrite insets : scale bars = 5 μm . B ) of occupied by surface Cav1 . 2 normalized to total ( GFP ) from non - permeabilized neurons transfected as in A ) . Data are represented as scattered dot plots with bar , mean ± S . D . ( n = 4 independent experiments ; Paired two - sample t - * p = 0 . 0318 ) .

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

A) Representative images of DIV 19 rat hippocampal neurons co-transfected with GFP (green channel) and Cav1.2-HA (red channel), together with either control or miR-499-5p mimics. After 12-13 days of expression, labeling with Anti-HA antibodies was performed in live conditions to identify cell surface Cav1.2 channels. Whole cells: scale bars = 20 μm; cell body and dendrite insets: scale bars= 5 μm. B) Quantification of the area occupied by surface Cav1.2 fluorescence normalized to the total cell area (GFP fluorescence) from non-permeabilized neurons transfected as in A). Data are represented as scattered dot plots with bar, mean±S.D. (n=4 independent experiments; Paired two-sample t-Test *p=0.0318).


D ) Representative Western blot of CACNB2 ( upper panel ) GAPDH ( lower panel ) expression in hippocampus of WT rats Cacna1c + / - rats injected with AAV - Control or AAV - miR - 499 hairpin . GAPDH was used as a loading control E ) of CACNB2 Western blots using hippocampal lysate from wild type rats injected with either AAV - control or AAV - miR - 499 . Data are represented as box plot with whiskers data points ( + : mean , : median ; whiskers : minimum maximum values ) . ( * p = 0 . 0103 , Unpaired two - sample t - ; n = 7 rats in AAV - Control group , n = 6 rats in AAV - miR - 499 group ) . F ) of CACNB2 Western blots using hippocampal lysate from Cacna1c + / - rats injected with either AAV - control or AAV - miR - 499 . Data are represented as box plot with whiskers data points ( + : mean , : median ; whiskers : minimum maximum values ) . ( * * p = 0 . 0044 , Unpaired two - sample t - ; n = 8 rats in AAV - Control group , n = 7 rats in AAV - miR - 499 group ) .

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

D) Representative Western blot images of CACNB2 (upper panel) and GAPDH (lower panel) protein expression levels in the hippocampus of WT rats and Cacna1c +/- rats injected with the AAV-Control or AAV-miR-499 hairpin viruses. GAPDH was used as a loading control E) Quantification of CACNB2 Western blots using hippocampal protein lysate from wild type rats injected with either AAV-control or AAV-miR-499. Data are represented as box plot with whiskers and data points (+: mean, line: median; whiskers: minimum and maximum values). (*p=0.0103, Unpaired two-sample t-Test; n=7 rats in the AAV-Control group, n=6 rats in the AAV-miR-499 group). F) Quantification of CACNB2 Western blots using hippocampal protein lysate from Cacna1c +/- rats injected with either AAV-control or AAV-miR-499. Data are represented as box plot with whiskers and data points (+: mean, line: median; whiskers: minimum and maximum values). (**p=0.0044, Unpaired two-sample t-Test; n=8 rats in the AAV-Control group, n=7 rats in the AAV-miR-499 group).


miR - 499 - 5p qPCR of total isolated from PBMCs of control ( = 26 , = 31 ) , BD ( = 26 , = 37 ) , or MDD ( = 18 , = 24 ) subjects . Wilcoxon rank sum after correction for antidepressant treatment ( linear model of form Fold Change ~ Group * Sex + + Antidepressant treatment ) . 2 - way ANOVA with correction for sex , antidepressant treatment ( linear model of form Fold Change ~ Group * Sex + + Antidepressant treatment ) . Control vs . BD : * * * * p = 8 . 89e - 07 ; control vs . MDD : n . s . p = 0 . 12674 . Data are presented as violin plots with median , quartiles data points .

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

miR-499-5p qPCR analysis of total RNA isolated from PBMCs of control (female= 26, male= 31), BD (female= 26, male= 37), or MDD (female= 18, male=24) subjects. Wilcoxon rank sum test after correction for age and antidepressant treatment (linear model of the form Fold Change ~ Group*Sex + Age + Antidepressant treatment). 2-way ANOVA with correction for sex, age and antidepressant treatment (linear model of the form Fold Change ~ Group*Sex + Age + Antidepressant treatment). Control vs. BD: ****p=8.89e-07; control vs. MDD: n.s. p=0.12674. Data are presented as violin plots with median, quartiles and data points.


miR - 499 - 5p qPCR of total isolated from PBMCs of control ( = 8 , = 10 ) SZ ( = 8 , = 15 ) subjects . 2 - way ANOVA , Main effect Sex : p = 0 . 9785 ; main effect Group : p = 0 . 0924 ; main effect Interaction : p = 0 . 9802 . Tukey ' s HSD : p = 0 . 0924 , ns . Data are presented as violin plots with median , quartiles data points .

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

miR-499-5p qPCR analysis of total RNA isolated from PBMCs of control (female= 8, male= 10) and SZ (female= 8, male= 15) subjects. 2-way ANOVA, Main effect Sex: p=0.9785; main effect Group: p= 0.0924; main effect Interaction: p= 0.9802. Tukey's HSD: p=0.0924, ns. Data are presented as violin plots with median, quartiles and data points.


miR - 499 - 5p qPCR of total isolated from PBMCs of control ( = 26 , = 31 ) , BD I ( = 9 , = 9 ) , or BDI II ( = 12 , = 16 ) subjects . 2 - way ANOVA , Main effect Sex : p = 0 . 174 ; main effect Group : * * * * p = 4 . 07e - 09 ; Interaction Group x Sex : p = 0 . 311 . Tukey ' s HSD : control vs . BDI : * * * * p = 0 . 0000037 . control vs . BDII : p = 0 . 0000002 . Data are presented as violin plots with median , quartiles data points .

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

miR-499-5p qPCR analysis of total RNA isolated from PBMCs of control (female= 26, male= 31), BD I (female= 9, male= 9), or BDI II (female= 12, male=16) subjects. 2-way ANOVA, Main effect Sex: p=0.174; main effect Group: ****p=4.07e-09; Interaction Group x Sex: p=0.311. Tukey's HSD: control vs. BDI: ****p=0.0000037. control vs. BDII:p=0.0000002. Data are presented as violin plots with median, quartiles and data points.


miR - 499 - 5p qPCR of total isolated from PBMCs of control subjects ( = 26 , = 31 ) , BD subjects in different mood states ( depressive ( = 7 , = 11 ) , euthymic ( = 7 , = 4 ) , hypomanic ( = 2 , = 6 ) , manic ( = 2 , = 1 ) , mixed ( = 1 , = 1 ) . 2 - way ANOVA , main effect Sex : p = 0 . 260 ; main effect Group : p = 1 . 26e - 07 ; Interaction Group x Sex : p = 0 . 059 ; Tukey ' s HSD : control vs . depressive * * * p = 0 . 0002621 , control vs . euthymic * * * p = 0 . 0008533 , control vs . hypomanic * * * * p = 0 . 0000470 . Data are presented as violin plots with median , quartiles data points .

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

miR-499-5p qPCR analysis of total RNA isolated from PBMCs of control subjects (female= 26, male= 31), and BD subjects in different mood states (depressive (female= 7, male=11), euthymic (female= 7, male=4), hypomanic (female=2, male=6), manic (female=2, male=1), mixed (female=1, male=1). 2-way ANOVA, main effect Sex: p=0.260; main effect Group: p=1.26e-07; Interaction Group x Sex: p=0.059; Tukey's HSD: control vs. depressive ***p=0.0002621, control vs. euthymic ***p=0.0008533, control vs. hypomanic ****p=0.0000470. Data are presented as violin plots with median, quartiles and data points.


miR - 499 - 5p qPCR of total isolated from PBMCs of healthy control subjects ( - CMT , n = 17 ) healthy subjects with a history of childhood maltreatment ( + CMT , n = 17 ) . ( * * * * p < 0 . 0001 , Mann - Whitney U ) . CMT : Childhood maltreatment . Data are presented as violin plots with median , quartiles data points .

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

miR-499-5p qPCR analysis of total RNA isolated from PBMCs of healthy control subjects (-CMT, n=17) and healthy subjects with a history of childhood maltreatment (+CMT, n=17). (****p<0.0001, Mann-Whitney U test). CMT: Childhood maltreatment. Data are presented as violin plots with median, quartiles and data points.


Association of miR - 499 - 5p expression GMV in HC ( n = 23 ) : miR - 499 - 5p were negatively correlated with a GMV cluster comprising parts of left Wernicke language ( i . e . transverse temporal gyrus , left parietal operculum left superior temporal gyrus ) ( in blue ) at p < . 05 cluster - level family wise error - corrected ( FWE ) for multiple comparisons after an initial threshold of p < . 001 uncorrected ( k = 1090 , x / y / z = - 42 / - 30 / 15 , t = 4 . 7 , p = . 045 ) .

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

Association of miR-499-5p expression and GMV in HC (n=23): miR-499-5p levels were negatively correlated with a GMV cluster comprising parts of the left Wernicke language area (i.e. transverse temporal gyrus, the left parietal operculum and the left superior temporal gyrus) (in blue) at p<.05 cluster-level family wise error-corrected (FWE) for multiple comparisons after an initial threshold of p<.001 uncorrected (k=1090, x/y/z=-42/-30/15, t= 4.7, p=.045).


( a ) Confocal time - lapse of an MCF10A - in - structure expressing GFP - LC3 H2B - mCherry . arrow marks recruitment of LC3 to entotic vacuole ( see Supplementary Movie S1 ) . Scale bar , 15 μm .

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

(a) Confocal time-lapse images of an MCF10A cell-in-cell structure expressing GFP-LC3 and H2B-mCherry. The arrow marks recruitment of LC3 to the entotic vacuole (see Supplementary Movie S1). Scale bar, 15 μm.


( b ) of MCF10A expressing GFP - LC3G120A mCherry - LC3 ; note no recruitment of GFP - LC3G120A to entotic vacuole . Scale bar , 15 μm . ( c ) LC3 is recruited from host - cytosol onto entotic vacuole . Data points are mean of GFP - LC3 on vacuoles versus cytoplasm for two independent - in - structures ( see Supplementary Movie S2 ) .

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

(b) Images of MCF10A cells expressing GFP-LC3G120A and mCherry-LC3; note no recruitment of GFP-LC3G120A to the entotic vacuole. Scale bar, 15 μm. (c) LC3 is recruited from the host-cell cytosol onto the entotic vacuole membrane. Data points are the mean fluorescence intensity of GFP-LC3 on vacuoles versus cytoplasm for two independent cell-in-cell structures (see Supplementary Movie S2).


( d ) Correlative video - - electron microscopy of an MCF10A GFP - LC3 - in - structure . were imaged by time - lapse microscopy , followed by fixation after LC3 recruitment ( see Supplementary Movie S3 ) . Top left , post - fixation of phase - contrast GFP - signals were taken demonstrating LC3 recruitment . Bottom left right , electron microscopy ( EM ) of same shows entotic vacuole is a single ( arrow in right image , which is a higher magnification of outlined in bottom left image )

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 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

(d) Correlative video-light-electron microscopy of an MCF10A GFP-LC3 cell-in-cell structure. Cells were imaged by time-lapse microscopy, followed by fixation after LC3 recruitment (see Supplementary Movie S3). Top left, post-fixation images of phase-contrast and GFP-fluorescence signals were taken demonstrating LC3 recruitment. Bottom left and right, electron microscopy (EM) of the same cell shows the entotic vacuole is a single membrane (arrow in right image, which is a higher magnification of the area outlined in the bottom left image)


( e ) of MCF10A - in - structures , with without autophagy inhibition , that show LC3 recruitment associated with of internalized . Data represent mean ± s . e . m . from at least three independent experiments ; n , total number of events ; * * * P0 . 0001 .

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

(e) Percentage of MCF10A cell-in-cell structures, with and without autophagy inhibition, that show LC3 recruitment associated with death of the internalized cell. Data represent mean±s.e.m. from at least three independent experiments; n, total number of death events; ***P0.0001.


( f , g ) Time - lapse of MCF10A - in - structures expressing either 2 × FYVE - mCherry ( red ) GFP - LC3 ( green ; f ) or Lamp1 - GFP ( green ) mCherry - LC3 ( red ; g ) . arrows indicate recruitment to entotic vacuole ( see Supplementary Movies S4 S5 ) . Times are indicated as h : min : s ( a , f ) or min ( g ) . Scale bars , 5 μm .

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 B-MEASURED_VAR O 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

(f,g) Time-lapse images of MCF10A cell-in-cell structures expressing either 2×FYVE-mCherry (red) and GFP-LC3 (green; f) or Lamp1-GFP (green) and mCherry-LC3 (red; g). The arrows indicate recruitment to the entotic vacuole (see Supplementary Movies S4 and S5). Times are indicated as h:min:s (a,f) or min (g). Scale bars, 5 μm.