Flow cytometric showing histograms MFI of O - propagyl - puromysin ( OPP ) incorporation in LT - HSC in : IF injection of EV from Molm - 14 , U - 937 , HL - 60 ( red , n = 5 , 43 ) , AML plasma ( Orange , n = 6 ) , or human CD34 + ( blue , n = 3 ) normalized to vehicle - injected femurs after subtracting background .

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

Flow cytometric analysis showing the histograms and MFI of O-propagyl-puromysin (OPP) incorporation in LT-HSC in: IF injection of EV from Molm-14, U-937, HL-60 (red, n=5,43), AML patients plasma (Orange, n=6), or human CD34+ cells (blue, n=3) normalized to vehicle-injected contralateral femurs after subtracting the background fluorescence.


qRT - PCR gene expression showing fold change of Pi3K / mTOR associated gene panel in KSL sorted from : ( A ) Molm - 14 - xenografted

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

qRT-PCR gene expression analysis showing the fold change of Pi3K/mTOR associated gene panel in KSL cells sorted from: (A) Molm-14-xenografted mice


qRT - PCR gene expression showing fold change of Pi3K / mTOR associated gene panel in KSL sorted from : IF injected mice with Molm - 14 - EV relative to their controls normalized to Gapdh endogenous control .

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

qRT-PCR gene expression analysis showing the fold change of Pi3K/mTOR associated gene panel in KSL cells sorted from: IF injected mice with Molm-14-EV relative to their controls and normalized to Gapdh endogenous control.


Flow cytometric showing histograms ( C )

Flow cytometric analysis showing the histograms (C)


Flow cytometric showing MFI of intracellular pS6KRP in LT - HSC in : ( D ) Molm - 14 - xenograft ( red , n = 9 ) vs . non - engrafted control ( black , n = 7 ) ,

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

Flow cytometric analysis showing the MFI of intracellular pS6KRP in LT-HSC in: (D) Molm-14-xenograft (red, n=9) vs. non-engrafted control (black, n=7),


Flow cytometric showing MFI of intracellular pS6KRP in LT - HSC in : IF injection of EV from Molm - 14 , U - 937 , HL - 60 ( red , n = 7 , 3 , 3 ) or human CD34 ( blue , n = 4 ) normalized to control femur with subtraction of background .

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Flow cytometric analysis showing the MFI of intracellular pS6KRP in LT-HSC in: IF injection of EV from Molm-14, U-937, HL-60 (red, n=7,3,3) or human CD34 cells (blue, n=4) normalized to the control contralateral femur with subtraction of background fluorescence.


Flow cytometric assessment of synthesis showing MFI of : ( F ) OPP , or ( G ) pS6RP in NIH - 3T3 72 hours after transfection with indicated miRNA mimics . results were calculated relative to control ( miR - scramble ) with background subtracted performed with at least three independent experiments , in technical replicates .

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

Flow cytometric assessment of protein synthesis showing the MFI of: (F) OPP, or (G) pS6RP in NIH-3T3 cells 72 hours after transfection with the indicated miRNA mimics. The results were calculated relative to control (miR-scramble) with the background fluorescence subtracted and performed with at least three independent experiments, in technical replicates.


Flow cytometric showing MFI of Raptor in NIH - 3T3 72 hours after transfection with indicated miRNA mimics . data were calculated relative to miR - scramble presented as means bars ± background was subtracted . Performed with at least three independent experiments , in technical replicates .

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Flow cytometric analysis showing the MFI of Raptor in NIH-3T3 cells 72 hours after transfection with the indicated miRNA mimics. The data were calculated relative to miR-scramble and presented as means bars ± SEM and the background fluorescence was subtracted. Performed with at least three independent experiments, in technical replicates.


Dual Luciferase reporter . NIH - 3T3 was transfected with miRNA mimics . Three hours later were transfected with Raptor 3 ′ UTR cloned into psiCheck - 2 vector for a total of 48 hours . Data are presented as % RLU ( relative luciferase units ) of miR - scramble control as means ± from at least three independent experiments , performed in technical replicates .

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

Dual Luciferase reporter assay. NIH-3T3 was transfected with the miRNA mimics. Three hours later the cells were transfected with the Raptor 3′UTR cloned into the psiCheck-2 vector for a total of 48 hours. Data are presented as %RLU (relative luciferase units) of the miR-scramble control as means ± SEM from at least three independent experiments, performed in technical replicates.


- cycle flow cytometric using Ki67 / Hoechst - 33342 of of LT - HSC in G0 phase after nucleofection of cKit + using AmaxaTM P3 4D - Nucleoeftcor kit , ( J ) were nucleofected with miR - Scramble ( CTRL ) or miR - 1246 mimic ( n = 3 ) for 72 hours ,

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

Cell-cycle flow cytometric analysis using Ki67/Hoechst-33342 staining of the percentage of LT-HSC in the G0 phase after nucleofection of cKit+ cells using the AmaxaTM P3 Primary Cell 4D-Nucleoeftcor kit, (J) Cells were nucleofected with miR-Scramble (CTRL) or miR-1246 mimic (n=3) for 72 hours,


- cycle flow cytometric using Ki67 / Hoechst - 33342 of of LT - HSC in G0 phase after nucleofection of cKit + using AmaxaTM P3 4D - Nucleoeftcor kit , were nucleofected with Anti - miR - Scramble ( CTRL ) or Anti - miR - 1246 ( n = 5 ) 1 - hour later co - treatment with Molm - 14 EVs for 72 hours .

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

Cell-cycle flow cytometric analysis using Ki67/Hoechst-33342 staining of the percentage of LT-HSC in the G0 phase after nucleofection of cKit+ cells using the AmaxaTM P3 Primary Cell 4D-Nucleoeftcor kit, Cells were nucleofected with Anti-miR-Scramble (CTRL) or Anti-miR-1246 (n=5) and 1-hour later co-treatment with Molm-14 EVs for 72 hours.


CFU - C showing relative number of colonies ( left panel ) colony subtypes ( right panel ) from KSL derived from Molm - 14 - xenografted mice ( red , n = 6 ) vs . non - engrafted control mice ( black , n = 5 ) in vitro cultured for 7 days in methylcellulose media . Experiments were performed in technical triplicates . " G " Granulocyte , " M " Monocyte , " E " Erythrocytes .

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

CFU-C assay showing the relative number of colonies (left panel) and the colony subtypes (right panel) from KSL cells derived from Molm-14-xenografted mice (red, n=6) vs. non-engrafted control mice (black, n=5) and in vitro cultured for 7 days in methylcellulose media. Experiments were performed in technical triplicates. "G" Granulocyte, "M" Monocyte, "E" Erythrocytes.


LT - HSC assessment after repopulation , recipient mice ( n = 4 per group ) were sacrificed after 21 days their LT - HSC were compared using P53 , pS6RP OPP flow cytometric assays . ( D - E ) Evaluation of in vivo long - term repopulation capacity .

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LT-HSC assessment after repopulation, recipient female mice (n=4 per group) were sacrificed after 21 days and their LT-HSC were compared using P53, pS6RP and OPP flow cytometric assays. (D-E) Evaluation of the in vivo long-term repopulation capacity.


Evaluation of in vivo long - term repopulation capacity . For secondary transplantation ( n = 6 per group ) , PB samples were collected twice weekly for 12 ( WK ) donor chimerism was measured by qPCR of Sex - determining Region Y ( SRY ) normalized to Gapdh gatekeeper as shown in left panel . Sixteen later , mice were sacrificed BM chimerism was assessed as shown in right panel . Tertiary transplantation , unfractionated BM from secondary recipients ( n = 6 per group , 106 per mouse ) were injected into tertiary 150cGy sub - lethally irradiated recipients blood SRY were assessed for 20 .

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Evaluation of the in vivo long-term repopulation capacity. For secondary transplantation (n=6 per group), PB samples were collected twice weekly for 12 weeks (WK) and the donor chimerism was measured by qPCR of Sex-determining Region Y (SRY) normalized to Gapdh gatekeeper as shown in the left panel. Sixteen weeks later, mice were sacrificed and BM male chimerism percentage was assessed as shown in the right panel. Tertiary transplantation, unfractionated BM cells from the secondary recipients (n=6 per group, 106 cells per mouse) were injected into tertiary 150cGy sub-lethally irradiated female recipients and blood SRY levels were assessed for 20 weeks.


Serial replating CFU - C presented as mean ± showing : ( B ) number of colonies per 1000 plated , ( C ) number of per colony , for each week time - point .

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

Serial replating CFU-C analysis presented as mean ± SEM showing: (B) the number of colonies per 1000 plated cells, and (C) the number of cells per colony, for each week time-point.


- damage assessment in KSL from recipients after two turns of replating . ( D , F , H ) Number of γH2AX foci in control - derived Molm - 14 - xenograft - derived : ( D ) recipient KSL , n = 211 321 , were stained with phosphoH2axser139 stain Hoechst ( blue ) , imaged using GE / API DV widefield microscope ( 60X objective ) to show γH2AX foci ( green ) counted using Imaris software .

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

DNA-damage assessment in KSL from primary recipients and after two turns of replating. (D, F, H) Number of γH2AX foci in control-derived and Molm-14-xenograft-derived: (D) Primary recipient KSL, n=211 and 321, All cells were stained with phosphoH2axser139 and the nuclear stain Hoechst (blue), imaged using the GE/API DV widefield microscope (60X objective) to show the γH2AX foci (green) and counted using Imaris software.


qRT - PCR gene expression showing fold - change of select - damage response - genes panel in Molm - 14 - xenograft - derived : ( E ) recipient KSL , n = 6 Data indicated by bars of fold - change calculated relative to matching controls , normalized to endogenous control Gapdh presented as means ± .

qRT-PCR gene expression analysis showing the fold-change of select DNA-damage response-genes panel in Molm-14-xenograft-derived: (E) Primary recipient KSL, n=6 Data indicated by bars of fold-change calculated relative to matching controls, normalized to the endogenous control Gapdh and presented as means ± SEM.


Number of γH2AX foci in control - derived Molm - 14 - xenograft - derived : from W1 plating , n = 218 369 were stained with phosphoH2axser139 stain Hoechst ( blue ) , imaged using GE / API DV widefield microscope ( 60X objective ) to show γH2AX foci ( green ) counted using Imaris software . Scale bars are 2μm .

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

Number of γH2AX foci in control-derived and Molm-14-xenograft-derived: cells from W1 plating, n=218 and 369 All cells were stained with phosphoH2axser139 and the nuclear stain Hoechst (blue), imaged using the GE/API DV widefield microscope (60X objective) to show the γH2AX foci (green) and counted using Imaris software. Scale bars are 2μm.


qRT - PCR gene expression showing fold - change of select - damage response - genes panel in Molm - 14 - xenograft - derived : from W1 plating , n = 4 , Data indicated by bars of fold - change calculated relative to matching controls , normalized to endogenous control Gapdh presented as means ± .

qRT-PCR gene expression analysis showing the fold-change of select DNA-damage response-genes panel in Molm-14-xenograft-derived: Cells from W1 plating, n=4, Data indicated by bars of fold-change calculated relative to matching controls, normalized to the endogenous control Gapdh and presented as means ± SEM.


Number of γH2AX foci in control - derived Molm - 14 - xenograft - derived : from W2 plating , n = 770 853 . were stained with phosphoH2axser139 stain Hoechst ( blue ) , imaged using GE / API DV widefield microscope ( 60X objective ) to show γH2AX foci ( green ) counted using Imaris software .

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

Number of γH2AX foci in control-derived and Molm-14-xenograft-derived: cells from W2 plating, n=770 and 853. All cells were stained with phosphoH2axser139 and the nuclear stain Hoechst (blue), imaged using the GE/API DV widefield microscope (60X objective) to show the γH2AX foci (green) and counted using Imaris software.


qRT - PCR gene expression showing fold - change of select - damage response - genes panel in Molm - 14 - xenograft - derived : from W2 plating n = 4 . Data indicated by bars of fold - change calculated relative to matching controls , normalized to endogenous control Gapdh presented as means ± .

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

qRT-PCR gene expression analysis showing the fold-change of select DNA-damage response-genes panel in Molm-14-xenograft-derived: Cells from W2 plating n=4. Data indicated by bars of fold-change calculated relative to matching controls, normalized to the endogenous control Gapdh and presented as means ± SEM.


umber of γH2AX foci , respectively , in control - Molm - 14 - xenograft - derived : ( K ) KSL , n = 101 / 204 , ( L ) LT - HSC , n = 33 / 53 . were processed similar to D - F . Scale bars are 3μm . Statistics : Student ' s t - . ( * * P < 0 . 01 ) .

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

umber of γH2AX foci, respectively, in control- and Molm-14-xenograft-derived: (K) KSL, n=101 / 204, and (L) LT-HSC, n=33 / 53. Images were processed similar to D-F. Scale bars are 3μm. Statistics: Student's t-test. (** P< 0.01).


qRT - PCR of - damage - genes panel in Molm - 14 - xenograft - derived : ( M ) KSL , ( N ) LT - HSC , relative to matched controls . Data depicted as fold - change relative to matching controls , normalized to endogenous control Gapdh presented as mean ± .

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

qRT-PCR analysis of the DNA-damage-genes panel in Molm-14-xenograft-derived: (M) KSL, and (N) LT-HSC, relative to matched controls. Data depicted as fold-change relative to matching controls, normalized to the endogenous control Gapdh and presented as mean ± SEM.


B ) , C ) Volcano plots showing fold change in average expression of phosphopeptides respectively , in CKO compared to WT ( q = Benjamini - Hochberg corrected p - value , n = 24 time points over 3 days ) . Statistically significant changes ( q ≤ 0 . 05 ) are shown in red . Some are labelled as space allows .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Volcano plots showing the fold change in average expression of all proteins and phosphopeptides respectively, in CKO cells compared to WT (q = Benjamini-Hochberg corrected p-value, n= 24 time points over 3 days). Statistically significant changes (q ≤ 0.05) are shown in red. Some proteins are labelled as space allows.


A ) Fold - change in relative amplitude ( RA ) was calculated for each of found to be rhythmic in both genotypes by RAIN ( i . e . no RA cut - off ) . mean fold change ( log ) in RA was an increase of 14 % in CKO compared to WT ( One sample t - , * * * * p < 0 . 0001 ) . B ) For each genotype , were divided into 10 deciles of equal number , ranked by abundance . mean abundance of each decile was plotted against proportion of decile that was rhythmic . Linear regression lines are shown for each genotype , slopes were significantly non - zero ( F , WT * * p = 0 . 0016 , CKO * * * * p < 0 . 0001 ) . slopes were also significantly different to each other ( F , p = 0 . 0001 ) . C ) Fold - change in relative amplitude ( RA ) was calculated for each of phosphopeptides found to be rhythmic in both genotypes by RAIN . On average , RA was increased in CKO compared to WT by 6 % ( One sample t - , * p < 0 . 05 ) . D ) same in B ) was carried out for phosphopeptides . slopes were significantly non - zero in CKO but not WT ( F , WT p = 0 . 3 ( ns . : non significant ) , CKO * p = 0 . 04 ) . slopes were also significantly different to each other ( F , p < 0 . 0001 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Fold-change in relative amplitude (RA) was calculated for each of the proteins found to be rhythmic in both genotypes by RAIN analysis (i.e. no RA cut-off). The mean fold change (log) in RA was an increase of 14% in CKO cells compared to WT cells (One sample t-test, ****p<0.0001). B) For each genotype, all proteins were divided into 10 deciles of equal number, ranked by abundance. The mean abundance of each decile was plotted against the proportion of the decile that was rhythmic. Linear regression lines are shown for each genotype, and the slopes were significantly non-zero (F test, WT **p=0.0016, CKO ****p<0.0001). The slopes were also significantly different to each other (F test, p=0.0001). C) Fold-change in relative amplitude (RA) was calculated for each of the phosphopeptides found to be rhythmic in both genotypes by RAIN. On average, the RA was increased in CKO cells compared to WT cells by 6% (One sample t-test, *p<0.05). D) The same analysis in B) was carried out for phosphopeptides. The slopes were significantly non-zero in CKO but not WT (F test, WT p=0.3 (ns.: non significant), CKO *p=0.04). The slopes were also significantly different to each other (F test, p<0.0001).


B ) Bar chart reports number % of detected showing a significant difference in abundance for listed comparisons by two - way ANOVA correcting for multiple comparisons , controlling for false discovery rate by Benjamini , Krieger Yekutieli method ( q = 0 . 05 ) .

O O O O O O O O O O O O O O O O O O O O O O O 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) Bar chart reports the number and % of detected proteins showing a significant difference in abundance for the listed comparisons by two-way ANOVA correcting for multiple comparisons, controlling for false discovery rate by the Benjamini, Krieger and Yekutieli method (q=0.05).


D - E ) with significant abundance change over time had no significant difference in mean absolute fold change between WT CKO ( D , p = 0 . 47 ) , but significantly greater variance in absolute fold change between independent CKO biological replicates than between their WT counterparts ( E , * * p = 0 . 0013 ) ; Kolmogorov - Smirmov ( n = 4 ) . Continuous lines = median , dotted lines = quartiles .

D-E) Proteins with significant abundance change over time had no significant difference in mean absolute fold change between WT and CKO cells (D, p=0.47), but significantly greater variance in absolute fold change between independent CKO biological replicates than between their WT counterparts (E, **p=0.0013); Kolmogorov-Smirmov test (n=4). Continuous lines = median, dotted lines = quartiles.


A ) From quantitative proteomics experiment , average abundance of catalytic proteasome subunits was calculated normalised to WT means . Trypsin - like ( β2 ) , chymotrypsin - like ( β3 ) caspase - like ( β1 ) catalytic subunits are shown . average was calculated from 24 time points of proteomics experiment . Mean ± SD , 2 - way ANOVA with Holm - Sidak ' s multiple comparisons , * * * * p ≤ 0 . 0001 .

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A) From the quantitative proteomics experiment, average abundance of catalytic proteasome subunits was calculated and normalised to WT means. Trypsin-like (β2), chymotrypsin-like (β3) and caspase-like (β1) catalytic subunits are shown. The average was calculated from all 24 time points of the proteomics experiment. Mean±SD, 2-way ANOVA with Holm-Sidak's multiple comparisons, ****p≤0.0001.


B ) Representative Western blot using an antibody that recognises 7 α subunits of 20S proteasome , with anti - histone H3 as loading control . C ) of blots in B ) , using replicates ( n = 3 ) , normalised to WT mean . Mean ± SD , one - Student ' s t with Welch correction - prior prediction from experiment in A ) that CKO abundance would be lower , * p ≤ 0 . 05 .

B) Representative Western blot using an antibody that recognises all 7 α subunits of the 20S proteasome, with anti-histone H3 as loading control. C) Quantification of the blots in B), using all replicates (n=3), normalised to WT mean. Mean±SD, one-tailed Student's t test with Welch correction - prior prediction from experiment in A) that CKO abundance would be lower, *p≤0.05.


D - E ) From one time - course experiment , ions , total were extracted in parallel samples . presented experiment is representative of 3 separate time - course experiments that were carried out ( N = 3 ) . Representative experiment shown ( n = 4 technical replicates for [ K + ] , n = 3 technical replicates for other traces ) . Blue lines highlight antiphasic relationship between oscillations in potassium . Mean ± , p - values from RAIN , red lines are fits by a damped cosine compared with a straight ( null hypothesis ) . Parallel PER2 : : LUC recordings were also performed plotted as a phase marker .

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D-E) From one time-course experiment, ions, cytosolic proteins and total protein were extracted in parallel samples. The presented experiment is representative of 3 separate time-course experiments that were carried out (N=3). Representative experiment shown (n=4 technical replicates for [K+], n=3 technical replicates for the other traces). Blue lines highlight the antiphasic relationship between oscillations in cytosolic protein and potassium concentration. Mean±SEM, p-values from RAIN, red lines are fits by a damped cosine compared with a straight line (null hypothesis). Parallel PER2::LUC recordings were also performed and plotted as a phase marker.


F ) Examples of key ion transporters are shown , as detected in proteomics experiment . P values show results of an F comparing fits of damped cosine against straight . except WT NKCC1 had RAIN p - values < 0 . 05 .

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F) Examples of key ion transporters are shown, as detected in the proteomics experiment. P values show the results of an F test comparing fits of damped cosine against straight line. All proteins except WT NKCC1 had RAIN p-values <0.05.


A ) WT CKO were treated with 500 nM tunicamycin ( TUN ) lysed in RIPA buffer at time points between 0 - 24 hours afterwards . Western blots were carried out , probing for phosphorylated eIF2α , total eIF2α actin . Representative blots shown , n = 4 . B ) of replicates represented in ( A ) , where phosphorylated eIF2α is normalised to total eIF2α . Mean ± SD . 2 - way ANOVA ( TWA ) with Holm - Sidak ' s multiple comparisons .

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

A) WT and CKO cells were treated with 500 nM tunicamycin (TUN) and lysed in RIPA buffer at time points between 0-24 hours afterwards. Western blots were carried out, probing for phosphorylated eIF2α, total eIF2α and actin. Representative blots shown, n=4. B) Quantification of all replicates represented in (A), where phosphorylated eIF2α is normalised to total eIF2α. Mean±SD. 2-way ANOVA (TWA) with Holm-Sidak's multiple comparisons.


C ) Mice were housed in 12h : 12h - cycles , with unrestricted feeding . Mouse lungs were collected 7 hours after transition to phase , 5 hours after intraperitoneal injection of mice with bortezomib ( BTZ , 2 . 5 mg / kg ) or vehicle ( Veh , 1 % DMSO in sterile PBS ) . Tissues were lysed in RIPA buffer western blots were performed , probing for phosphorylated STAT3 , total STAT3 actin . ( left ) ( right ) replicates shown , N = 2 mice for each condition . D ) of blots shown in ( C ) , where phosphorylated STAT3 is normalised to total STAT3 . Mean ± SD . Multiple t - , corrected for multiple comparisons with Holm - Sidak method , * p ≤ 0 . 05 , * * * p ≤ 0 . 001 .

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

C) Mice were housed in 12h:12h light-dark cycles, with unrestricted feeding. Mouse lungs were collected 7 hours after transition to the light phase, 5 hours after intraperitoneal injection of mice with bortezomib (BTZ, 2.5 mg/kg) or vehicle (Veh, 1% DMSO in sterile PBS). Tissues were lysed in RIPA buffer and western blots were performed, probing for phosphorylated STAT3, total STAT3 and actin. Male (left) and female (right) replicates shown, N=2 mice for each condition. D) Quantification of the blots shown in (C), where phosphorylated STAT3 is normalised to total STAT3. Mean±SD. Multiple t-tests, corrected for multiple comparisons with the Holm-Sidak method, *p≤0.05, ***p≤0.001.


A ) WT PER2 : : LUC were treated with epoxomicin or vehicle control . Blue arrows show time points where drug was washed off . Mean ( solid lines ) ± SD ( dotted lines ) . B ) of damping rate of PER2 : : LUC recordings shown in A ) , for duration of drug treatment . Mean ± SD . One - way ANOVA with Holm - Sidak ' s multiple comparisons , * * p ≤ 0 . 01 , * * * * p ≤ 0 . 0001 . C ) WT PER2 : : LUC were treated with tunicamycin or vehicle control . Mean ( solid lines ) ± SD ( dotted lines ) . D ) of damping rate of PER2 : : LUC recordings shown in C ) , for duration of drug treatment . Mean ± SD . One - way ANOVA with Holm - Sidak ' s multiple comparisons , * * * * p ≤ 0 . 0001 .

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 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 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 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

A) WT PER2::LUC cells were treated with epoxomicin or vehicle control. Blue arrows show the time points where drug was washed off. Mean (solid lines) ± SD (dotted lines). B) Quantification of damping rate of the PER2::LUC recordings shown in A), for the duration of drug treatment. Mean±SD. One-way ANOVA with Holm-Sidak's multiple comparisons, **p≤0.01, ****p≤0.0001. C) WT PER2::LUC cells were treated with tunicamycin or vehicle control. Mean (solid lines) ± SD (dotted lines). D) Quantification of damping rate of the PER2::LUC recordings shown in C), for the duration of drug treatment. Mean±SD. One-way ANOVA with Holm-Sidak's multiple comparisons, ****p≤0.0001.


E ) WT PER2 : : LUC were treated with radicicol or vehicle control . Blue arrows show time points where drug was washed off . Mean ( solid lines ) ± SD ( dotted lines ) . F ) of damping rate of PER2 : : LUC recordings shown in E ) , for duration of drug treatment . Mean ± SD . One - way ANOVA with Holm - Sidak ' s multiple comparisons , * * * * p ≤ 0 . 0001 .

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E) WT PER2::LUC cells were treated with radicicol or vehicle control. Blue arrows show the time points where drug was washed off. Mean (solid lines) ± SD (dotted lines). F) Quantification of damping rate of the PER2::LUC recordings shown in E), for the duration of drug treatment. Mean±SD. One-way ANOVA with Holm-Sidak's multiple comparisons, ****p≤0.0001.


I - J ) Representative double - plotted actograms of WT mice receiving drinking water with DMSO vehicle control ( N = 7 mice ) or 35 µg / mL ixazomib ( N = 8 mice ) . Two representative actograms are shown per condition . For first 7 days mice were in 12 : 12h - cycles ( LD ) , had drinking water supplemented with blackcurrant squash . Ixazomib / DMSO was added to this , lighting was changed to constant darkness ( DD ) . After 7 days drug / vehicle was removed , mice remained in DD until end of experiment .

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I-J) Representative double-plotted actograms of WT mice receiving drinking water with DMSO vehicle control (N=7 mice) or 35 µg/mL ixazomib (N=8 mice). Two representative actograms are shown per condition. For the first 7 days the mice were in 12:12h light-dark cycles (LD), and had drinking water supplemented with blackcurrant squash. Ixazomib/DMSO was added to this, and the lighting was changed to constant darkness (DD). After 7 days the drug/vehicle was removed, and the mice remained in DD until the end of the experiment.


A . Bclaf1 wild type ( WT ) two Bclaf1 knockout HeLa lines ( KO - 1 - 2 ) were pre - treated with DMSO or cycloheximide ( CHX , 1 μg / ml ) for 30 min , then treated with TNF ( 10 ng / ml ) together with CHX for 12 hours . Afterwards , were subjected to Annexin V / 7AAD followed by flow cytometry , Annexin V positive were counted for , or extractions followed by western blotting . B , C . HeLa ( B ) or MEFs ( C ) were transfected with control ( siCtrl ) or two siRNAs against Bclaf1 ( siBclaf1 - 1 - 2 ) then treated analyzed as described in A . Data information : Data are shown as mean ± SD . n = 3 biological replicates . ns , not significant ; * * p < 0 . 01 ; * * * * p < 0 . 0001 . One - way ANOVA .

O O B-CONTROLLED_VAR 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 B-MEASURED_VAR I-MEASURED_VAR 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 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

A. Bclaf1 wild type (WT) and two Bclaf1 knockout HeLa cell lines (KO-1 and -2) were pre-treated with DMSO or cycloheximide (CHX, 1 μg/ml) for 30 min, and then treated with TNF (10 ng/ml) together with CHX for 12 hours. Afterwards, the cells were subjected to Annexin V/7AAD staining followed by flow cytometry analysis, all Annexin V positive cells were counted for analysis, or protein extractions followed by western blotting analysis. B, C. HeLa (B) or primary MEFs (C) were transfected with control (siCtrl) or two siRNAs against Bclaf1 (siBclaf1-1 and -2) and then treated and analyzed as described in A. Data information: Data are shown as mean ± SD. n=3 biological replicates. ns, not significant; **p<0.01; ****p<0.0001. One-way ANOVA test.


D , E . MEFs transfected with control ( siCtrl ) or siBclaf1 - 1 / or siBclaf1 - 2 siRNAs were pre - treated with CHX ( 1 μg / ml ) / z - VAD ( 20 μM ) ( D ) or SM - 164 ( 100 nM ) / z - VAD ( 20 μM ) ( E ) for 30 min , then added TNF ( 10 ng / ml ) for indicated times . were subjected to Annexin V / 7AAD after 9 hours followed by flow cytometry for 7AAD positive Annexin V negative ( up ) , or extractions western blotting at indicated time periods ( down ) . Data information : Data are shown as mean ± SD . n = 3 biological replicates . ns , not significant ; * * p < 0 . 01 ; * * * * p < 0 . 0001 . One - way ANOVA .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 B-MEASURED_VAR I-MEASURED_VAR 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 O O O O O O O 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. MEFs transfected with control (siCtrl) or siBclaf1-1 and/or siBclaf1-2 siRNAs were pre-treated with CHX (1 μg/ml)/z-VAD (20 μM) (D) or SM-164 (100 nM)/z-VAD (20 μM) (E) for 30 min, and then added TNF (10 ng/ml) for indicated times. The cells were subjected to Annexin V/7AAD staining after 9 hours followed by flow cytometry analysis for 7AAD positive and Annexin V negative cells (up), or protein extractions and western blotting analysis at indicated time periods (down). Data information: Data are shown as mean ± SD. n=3 biological replicates. ns, not significant; **p<0.01; ****p<0.0001. One-way ANOVA test.


A , B . lysates of HeLa Bclaf1 WT KO ( A ) , HeLa transfected with siCtrl or siBclaf1 - 1 ( B ) treated with TNF ( 10 ng / ml ) for indicated time periods were analyzed by western blotting with indicated antibodies .

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A, B. Cell lysates of HeLa Bclaf1 WT and KO cells (A), and HeLa cells transfected with siCtrl or siBclaf1-1 (B) treated with TNF (10 ng/ml) for indicated time periods were analyzed by western blotting with the indicated antibodies.


C , D . HeLa Bclaf1 WT KO ( C ) , HeLa transfected with siCtrl or siBclaf1 - 1 ( D ) were treated with TNF ( 10 ng / ml ) for indicated time periods , then subject to nucleus - cytoplasm fractionation as described in materials methods followed by western blotting . Tubulin histone H3 were used as loading controls for fractions , respectively .

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C, D. HeLa Bclaf1 WT and KO cells (C), and HeLa cells transfected with siCtrl or siBclaf1-1 (D) were treated with TNF (10 ng/ml) for the indicated time periods, and then subject to nucleus-cytoplasm fractionation as described in materials and methods followed by western blotting. Tubulin and histone H3 were used as loading controls for cytoplasmic and nuclear fractions, respectively.


B . MEFs treated with TNF ( 10 ng / ml ) for 4 hours were subject to FADD immunoprecipitation , then analyzed by western blotting .

O O O O O B-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

B. MEFs treated with TNF (10 ng/ml) for 4 hours were subject to FADD immunoprecipitation, and then analyzed by western blotting.


G , H . HeLa transfected with siRNAs against Bclaf1 or c - FLIP were pretreated with CHX ( 1 μg / ml ) for 30 min then stimulated with TNF ( 10 ng / ml ) in presence of CHX for 12 hours followed by Annexin V / 7AAD flow cytometry ( G ) , or extractions followed by western blotting ( H ) . Data information : Data are shown as mean ± SD . n = 3 biological replicates . * * p < 0 . 01 ; * * * * p < 0 . 0001 . One - way ANOVA .

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

G, H. HeLa cells transfected with siRNAs against Bclaf1 or c-FLIP were pretreated with CHX (1 μg/ml) for 30 min and then stimulated with TNF (10 ng/ml) in the presence of CHX for 12 hours followed by Annexin V/7AAD staining and flow cytometry analysis (G), or protein extractions followed by western blotting analysis (H). Data information: Data are shown as mean ± SD. n=3 biological replicates. **p<0.01; ****p<0.0001. One-way ANOVA test.


A - F . Bclaf1 WT , KO - 1 - 2 HeLa ( A D ) , as well as HeLa ( B E ) MEFs ( C F ) which were transfected with siCtrl or siBclaf1 - 1 - 2 were treated with TNF ( 10 ng / ml ) for 6 hours followed by western blotting ( A - C ) , or for 4 hours followed by total extraction RT - PCR ( D - F ) . Data information : Data are shown as mean ± SD . n = 3 biological replicates . * p < 0 . 05 ; * * * p < 0 . 001 ; * * * * p < 0 . 0001 . One - way ANOVA .

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

A-F. Bclaf1 WT, KO-1 and -2 HeLa cells (A and D), as well as HeLa (B and E) and primary MEFs (C and F) which were transfected with siCtrl or siBclaf1-1 and -2 were treated with TNF (10 ng/ml) for 6 hours followed by western blotting analysis (A-C), or for 4 hours followed by total RNA extraction and RT-PCR analysis (D-F). Data information: Data are shown as mean ± SD. n=3 biological replicates. *p<0.05; ***p<0.001; ****p<0.0001. One-way ANOVA test.


H , I . CHIP of binding of Bclaf1 to promoter of CFLAR was performed in HeLa - Flag - Bclaf1 treated without ( H ) or with TNF ( I ) . regions amplified by RT - PCR were delineated . Data information : Data are shown as mean ± SD . n = 3 biological replicates . * p < 0 . 05 ; * * * p < 0 . 001 ; * * * * p < 0 . 0001 . One - way ANOVA .

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H, I. CHIP analysis of the binding of Bclaf1 to the promoter of CFLAR was performed in HeLa-Flag-Bclaf1 cells treated without (H) or with TNF (I). The regions amplified by RT-PCR were delineated. Data information: Data are shown as mean ± SD. n=3 biological replicates. *p<0.05; ***p<0.001; ****p<0.0001. One-way ANOVA test.


B . CFLAR promoter were co - transfected with Bclaf1 or / p50 into HeLa - Bclaf1 - KO , luciferase activity was measured as described before .

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B. CFLAR promoter were co-transfected with Bclaf1 or/and p50 into HeLa-Bclaf1-KO cells, and the luciferase activity was measured as described before.


E , HeLa were transfected with siCtrl or siRNA against Bclaf1 or p50 were pre - treated with DMSO or CHX ( 1 μg / ml ) for 30 min , then treated with TNF ( 10 ng / ml ) for 12 hours . were lysed then analyzed by western

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E, HeLa cells were transfected with siCtrl or siRNA against Bclaf1 or p50 were pre-treated with DMSO or CHX (1 μg/ml) for 30 min, and then treated with TNF (10 ng/ml) for 12 hours. Cells were lysed and then analyzed by western blotting


G . CHIP of CFLAR promoter in HeLa - Flag - Bclaf1 . Immunoprecipitation was performed using anti - Flag control IgG antibodies followed by RT - PCR . Data information : Data are shown as mean ± SD . n = 3 biological replicates . ns , not significant ; * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 ; * * * * p < 0 . 0001 . One - way or Two - way ANOVA .

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G. CHIP analysis of the CFLAR promoter in HeLa-Flag-Bclaf1 cells. Immunoprecipitation was performed using anti-Flag and control IgG antibodies followed by RT-PCR analysis. Data information: Data are shown as mean ± SD. n=3 biological replicates. ns, not significant; *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001. One-way or Two-way ANOVA test.


A . HeLa - Flag - Bclaf1 Bclaf1 WT HeLa treated with or without TNF ( 10 ng / ml ) for 2 hours were separated into fraction . Endogenous Flag - Bclaf1 in each fraction was immunoprecipitated with M2 beads analyzed by western blotting .

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A. HeLa-Flag-Bclaf1 and Bclaf1 WT HeLa cells treated with or without TNF (10 ng/ml) for 2 hours were separated into the cytoplasmic and the nuclear fraction. Endogenous Flag-Bclaf1 complex in each fraction was immunoprecipitated with M2 beads and analyzed by western blotting.


B . HeLa - Flag - Bclaf1 transfected with siCtrl or sip65 were treated with TNF ( 10 ng / ml ) for 2 hours . fraction was isolated subjected to immunoprecipitation with an anti - Flag or control IgG antibody , followed by western blotting .

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B. HeLa-Flag-Bclaf1 cells transfected with siCtrl or sip65 were treated with TNF (10 ng/ml) for 2 hours. The nuclear fraction was isolated and subjected to immunoprecipitation with an anti-Flag or control IgG antibody, followed by western blotting analysis.


D . CFLAR FL promoter were co - transfected with p50 Bclaf1 FL or its deletion mutant into HeLa - Bclaf1 - KO . experiments were performed in triplicate with a Renilla reporter in transfection mixture for normalization . Data information : Data are shown as mean ± SD . n = 3 biological replicates . ns , not significant . One - way ANOVA .

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D. CFLAR FL promoter were co-transfected with p50 and Bclaf1 FL or its individual deletion mutant into HeLa-Bclaf1-KO cells. The experiments were performed in triplicate with a Renilla reporter in the transfection mixture for normalization. Data information: Data are shown as mean ± SD. n=3 biological replicates. ns, not significant. One-way ANOVA test.


siRNAs against Bclaf1 control siRNAs ( siCtrl ) mixed with in vivo - jetPEI ( Polyplus ) were injected into C57BL / 6 mice . mice were then treated with mTNF for two hours before sacrifice . small intestines were excised processed for immunohistochemical for Bclaf1 ( B ) Hematoxylin - - eosin ( C ) Bclaf1 knockdown efficiency is detected by immunohistochemical ( B ) . Hematoxylin - - eosin showing morphological change ( C ) . Villus height crypt depth were measured ( D ) . Data are shown as mean ± SD . n = 5 mice for each group . ns , not significant ; * * p < 0 . 01 ; * * * p < 0 . 001 ; * * * * p < 0 . 0001 . One - way ANOVA . Scale bars : 50 μm in B , 100 μm in C .

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siRNAs against Bclaf1 and control siRNAs (siCtrl) mixed with in vivo-jetPEI (Polyplus) were injected into C57BL/6 mice. The mice were then treated with mTNF for two hours before sacrifice. The small intestines were excised and processed for immunohistochemical staining for Bclaf1 (B) and Hematoxylin-and-eosin staining (C) Bclaf1 knockdown efficiency is detected by immunohistochemical analysis (B). Hematoxylin-and-eosin staining showing the morphological change (C). Villus height and crypt depth were measured (D). Data are shown as mean ± SD. n=5 mice for each group. ns, not significant; **p<0.01; ***p<0.001; ****p<0.0001. One-way ANOVA test. Scale bars: 50 μm in B, 100 μm in C.


siRNAs against Bclaf1 control siRNAs ( siCtrl ) mixed with in vivo - jetPEI ( Polyplus ) were injected into C57BL / 6 mice . mice were then treated with mTNF for two hours before sacrifice . small intestines were excised processed for immunohistochemical for caspase 3 ( E ) , phospholated RIPK3 ( F ) CC3 + p - RIPK3 + positive in five fields per intestine were quantified . Data are shown as mean ± SD . n = 5 mice for each group . ns , not significant ; * * p < 0 . 01 ; * * * p < 0 . 001 ; * * * * p < 0 . 0001 . One - way ANOVA . Scale bars : 50

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

siRNAs against Bclaf1 and control siRNAs (siCtrl) mixed with in vivo-jetPEI (Polyplus) were injected into C57BL/6 mice. The mice were then treated with mTNF for two hours before sacrifice. The small intestines were excised and processed for immunohistochemical staining for cleaved caspase 3 (E), phospholated RIPK3 (F) CC3+ and p-RIPK3+ positive cells in five fields per intestine were quantified. Data are shown as mean ± SD. n=5 mice for each group. ns, not significant; **p<0.01; ***p<0.001; ****p<0.0001. One-way ANOVA test. Scale bars: 50 μm


siRNAs against Bclaf1 control siRNAs ( siCtrl ) mixed with in vivo - jetPEI ( Polyplus ) were injected into C57BL / 6 mice . mice were then treated with mTNF for two hours before sacrifice . small intestines were excised processed for western blotting ( G ) .

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siRNAs against Bclaf1 and control siRNAs (siCtrl) mixed with in vivo-jetPEI (Polyplus) were injected into C57BL/6 mice. The mice were then treated with mTNF for two hours before sacrifice. The small intestines were excised and processed for western blotting analysis (G).


HIEC - 6 were transfected siCtrl or siBclaf1 - 1 - 2 . Then were treated with TNF for 24 hours were subjected for extractions western blotting ( I J )

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HIEC-6 cells were transfected siCtrl or siBclaf1-1 and -2. Then the cells were treated with TNF for 24 hours were subjected for protein extractions and western blotting analysis (I and J)


K . Representative immunostainings for Pax7 MyoD in eMPs plated on Vtn maintained in proliferation media ( PM ) after isolation . L . Representative immunostainings for Pax7 MyoD in eMPs plated on Vtn that were first maintained in PM then switched to differentiation media ( DM ) . hiPSC donor 1 .

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K. Representative immunostainings for Pax7 and MyoD in eMPs plated on Vtn and maintained in proliferation media (PM) after isolation. L. Representative immunostainings for Pax7 and MyoD in eMPs plated on Vtn that were first maintained in PM and then switched to differentiation media (DM). hiPSC donor 1.


M - O . of number of Pax7 ( M ) , MyoD ( N ) Pax7 / MyoD double - positive ( O ) in hskMPs eMPs in PM after switching to DM . hiPSC donor 1 .

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M-O. Quantification of the number of Pax7 (M), MyoD (N) and Pax7/MyoD double-positive (O) cells in hskMPs and eMPs in PM and after switching to DM. hiPSC donor 1.


P . Representative immunostaining for muscle fiber marker myosin heavy chain ( MyHC ) of eMPs maintained in DM . Q . of fusion index of hskMPs eMPs in PM DM . hiPSC donor 1 .

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P. Representative immunostaining for the muscle fiber marker myosin heavy chain (MyHC) of eMPs maintained in DM. Q. Quantification of the fusion index of hskMPs and eMPs in PM and DM. hiPSC donor 1.


U - Z , Representative immunostainings of eMPs in DM expressing Pax7 ( V ) , MyoD ( X ) , MyoG ( Z ) , as well as proliferation marker Ki67 . hiPSC donor 1 .

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U-Z, Representative immunostainings and quantification of eMPs in DM expressing Pax7 (V), MyoD (X), and MyoG (Z), as well as the proliferation marker Ki67. hiPSC donor 1.


A . Representative immunostainings for dystrophin in cross - sections of of mdx mice after transplantation of hskMPs or eMPs . Scale bars in two left - side = 300 μm . Scale bars in inserts 1 2 = 50 μm . B . Number of dystrophin positive in mdx injected with vehicle ( Ctrl ) , eMPs hskMPs . C . Maximal of clusters of dystrophin positive in cross - sections of mdx transplanted with eMPs hskMPs . A - C . 10 days post - transplantation . hiPSC donor 1 .

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A. Representative immunostainings for dystrophin in cross-sections of muscles of mdx mice after transplantation of hskMPs or eMPs. Scale bars in the two left-hand side images = 300 μm. Scale bars in inserts 1 and 2 = 50 μm. B. Number of dystrophin positive fibers in mdx muscles injected with vehicle (Ctrl), eMPs and hskMPs. C. Maximal distance of clusters of dystrophin positive fibers in cross-sections of mdx muscles transplanted with eMPs and hskMPs. A-C. 10 days post-transplantation. hiPSC donor 1.


( a - c ) Low - magnification cryo - EM projection of pig ( a ) , horse ( b ) , mouse ( c ) sperm . Different regions of flagellum are annotated as follows : green - neck , yellow - midpiece , coral - principal piece , pink - endpiece . ( d - f ) Tomographic slices through cryo - FIB milled lamellae of pig ( d ) , horse ( e ) , mouse ( f ) sperm . Transverse slices ( d ' - f ' ) show complete triplets in pig ( d ' ) horse ( e ' ) , but not in mouse ( f ' ) . Complete triplets are indicated by green arrowheads with black outlines , while degenerated triplets are indicated by arrowheads with green outlines . Note electron - dense material in lumen of pig sperm proximal centriole that is continuous with connecting piece ( asterisks in d d ' ) .

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(a-c) Low-magnification cryo-EM projection images of pig (a), horse (b), and mouse (c) sperm. Different regions of the flagellum are annotated as follows: green - neck, yellow - midpiece, coral - principal piece, pink - endpiece. (d-f) Tomographic slices through cryo-FIB milled lamellae of pig (d), horse (e), and mouse (f) sperm. Transverse slices (d'-f') show complete triplets in the pig (d') and the horse (e'), but not in the mouse (f'). Complete triplets are indicated by green arrowheads with black outlines, while degenerated triplets are indicated by white arrowheads with green outlines. Note the electron-dense material in the lumen of the pig sperm proximal centriole that is continuous with the connecting piece (asterisks in d and d').


( g - i ) Tomographic slices through cryo - FIB milled lamellae of DC - to - axoneme transition in pig sperm show how change in geometry ( g , arrows dashed lines ) coincides with appearance of axoneme accessory structures ( h , arrows ) with density in A - tubule ( i , compare insets in black boxes ) . In ( i ) , black arrows indicate where cross - sections in black boxes were taken from .

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(g-i) Tomographic slices through cryo-FIB milled lamellae of the DC-to-axoneme transition in pig sperm show how the change in geometry (g, white arrows and white dashed lines) coincides with the appearance of axoneme accessory structures (h, white arrows) and with density in the A-tubule (i, compare insets in white and black boxes). In (i), the white and black arrows indicate where the cross-sections in white and black boxes were taken from.


( a - c ) Slices through defocus cryo - tomograms of endpiece in pig ( a ) , horse ( b ) , mouse ( c ) sperm . Insets show digital zooms ( black boxes ) subtomogram averages ( boxes ) of microtubule tips . Note presence of a plug ( arrowhead ) extending into lumen .

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(a-c) Slices through defocus cryo-tomograms of the endpiece in pig (a), horse (b), and mouse (c) sperm. Insets show digital zooms (black boxes) and subtomogram averages (white boxes) of microtubule tips. Note the presence of a plug (arrowhead) extending into the lumen.


A FACS profiles of content for arrested at different points in cycle by Dup / Cdt1 RNAi , cyclin E RNAi , Cdk2 RNAi , Dacapo overexpression ( + Dacapo ) , 1 mM HU .

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A FACS profiles of DNA content for cells arrested at different points in the cell cycle by Dup/Cdt1 RNAi, cyclin E RNAi, Cdk2 RNAi, Dacapo overexpression (+Dacapo), and 1 mM HU.


B of Mcm2 ‐ 7 association at different points in cycle . fractions extracts were assayed for Orc2 Mcm2 ‐ 7 by Western blot . A non ‐ specific band is indicated by an asterisk ( * ) . C of of ‐ bound Mcm2 ‐ 7 relative to Orc2 ( log10 scale ) for a minimum of 11 replicates ( mean ± SD ) .

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B Analysis of Mcm2‐7 nuclear chromatin association at different points in the cell cycle. Nuclear chromatin fractions and cytoplasmic extracts were assayed for Orc2 and Mcm2‐7 by Western blot. A non‐specific band is indicated by an asterisk (*).C Quantification of the ratio of chromatin‐bound Mcm2‐7 relative to Orc2 (log10 scale) for a minimum of 11 replicates (mean ± SD).


cycle by FACS of arrested in G1 by Dacapo overexpression followed by release back into cycle for 13 h ( top ) . Western blot of Mcm2 ‐ 7 Orc2 for fractions of Mcm2 ‐ 7 , Orc2 , Dacapo .

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Cell cycle analysis by FACS of cells arrested in G1 by Dacapo overexpression followed by release back into the cell cycle for 13 h (top). Western blot analysis of Mcm2‐7 and Orc2 for the nuclear chromatin and cytoplasmic fractions of Mcm2‐7, Orc2, and Dacapo.


B FACS profiles of content for each population assayed .

B FACS profiles of DNA content for each cell population assayed.


C Western blot for Orc2 Mcm2 ‐ 7 in fraction Orc2 , Mcm2 ‐ 7 , Dup / Cdt1 , cyclin E in fraction for each condition assayed .

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C Western blot analysis for Orc2 and Mcm2‐7 in the nuclear chromatin fraction and Orc2, Mcm2‐7, Dup/Cdt1, and cyclin E in the cytoplasmic fraction for each condition assayed.


A Genome ‐ wide of ORC localization by ChIP ‐ chip . ORC enrichment from asynchronous is depicted for a 5 ‐ Mb section of chromosome 2L . B Genome ‐ wide of Mcm2 ‐ 7 localization in early G1 by ChIP ‐ chip . Mcm2 ‐ 7 enrichment from cyclin E RNAi ‐ depleted is depicted for a 5 ‐ Mb section of chromosome 2L . C Venn diagram depicting overlap between ORC Mcm2 ‐ 7 peaks .

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A Genome‐wide analysis of ORC localization by ChIP‐chip. ORC enrichment from asynchronous cells is depicted for a 5‐Mb section of chromosome 2L.B Genome‐wide analysis of Mcm2‐7 localization in early G1 by ChIP‐chip. Mcm2‐7 enrichment from cyclin E RNAi‐depleted cells is depicted for a 5‐Mb section of chromosome 2L.C Venn diagram depicting the overlap between ORC and Mcm2‐7 peaks.


A Genome ‐ wide of Mcm2 ‐ 7 localization at G1 / S transition by ChIP ‐ chip . Mcm2 ‐ 7 enrichment from HU ‐ arrested is depicted for a 5 ‐ Mb section of chromosome 2L . Inset : transcribed ( green ) non ‐ transcribed ( red ) genes are indicated above with genes on positive strand on top those on negative on bottom . B Bimodal distribution of Mcm2 ‐ 7 enrichment over transcribed non ‐ transcribed genes . Histogram showing distribution of probe scores found within transcribed ( green ) non ‐ transcribed ( red ) genes . C " Meta " ‐ gene of Mcm2 ‐ 7 enrichment for different deciles of gene expression their aggregated probe intensities .

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A Genome‐wide analysis of Mcm2‐7 localization at the G1/S transition by ChIP‐chip. Mcm2‐7 enrichment from HU‐arrested cells is depicted for a 5‐Mb section of chromosome 2L. Inset: transcribed (green) and non‐transcribed (red) genes are indicated above with genes on the positive strand on the top and those on the negative on the bottom.B Bimodal distribution of Mcm2‐7 enrichment over transcribed and non‐transcribed genes. Histogram showing the distribution of probe scores found within transcribed (green) and non‐transcribed (red) genes.C "Meta"‐gene analysis of Mcm2‐7 enrichment for different deciles of gene expression and their aggregated probe intensities.


D Mcm2 ‐ 7 is displaced from by replication . Genome ‐ wide of Mcm2 ‐ 7 localization in late S ‐ phase ( 6 h post HU release ) by ChIP ‐ chip . Mcm2 ‐ 7 enrichment is depicted for a 10 ‐ Mb section of chromosome 2L ( filled gray ) , replication timing profile ( black ) early ( yellow ) late ( purple ) replication timing domains . E Box ‐ plots representing late S ‐ phase Mcm2 ‐ 7 ChIP signal found within early ( 246 ) or late ( 167 ) replication domains .

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D Mcm2‐7 is displaced from chromatin by DNA replication. Genome‐wide analysis of Mcm2‐7 localization in late S‐phase (6 h post HU release) by ChIP‐chip. Mcm2‐7 enrichment is depicted for a 10‐Mb section of chromosome 2L (filled gray), replication timing profile (black line) and early (yellow) and late (purple) replication timing domains.E Box‐plots representing late S‐phase Mcm2‐7 ChIP signal found within early (246) or late (167) replication domains.


A . Ovaries from control heterozygous Rpp3018 . 2 flies ( left ) , homozygous Rpp3018 . 2 or transheterozygous Rpp3018 . 2 / Rpp30PE flies ( middle ) homozygous Rpp3018 . 2 carrying a copy of ubiRpp30GFP transgene ( right ) . Scale bar , 100µm .

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A. Ovaries from control heterozygous Rpp3018.2 flies (left), homozygous Rpp3018.2 or transheterozygous Rpp3018.2/Rpp30PE flies (middle) and homozygous Rpp3018.2 carrying a copy of ubiRpp30GFP transgene (right). Scale bar, 100µm.


D . Ovaries were dissected , lysed extracts analyzed by Western Blot using anti - Rpp30 , anti - GFP or anti - tubulin antibodies . Orange arrows indicate overexpressed endogenous Rpp30 signals . Genotypes are numbered as 1 , 2 , 3 .

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D. Ovaries were dissected, lysed and protein extracts analyzed by Western Blot using anti-Rpp30, anti- GFP or anti-tubulin antibodies. Orange arrows indicate overexpressed and endogenous Rpp30 signals. Genotypes are numbered as 1,2,3.


E . Rpp30 - dependent early oogenesis arrest is restored by expression of Rpp30GFP transgene in two different genetic backgrounds : Left . Homozygous Rpp3018 . 2 ovariole expressing ubiquitous ubiRpp30GFP ( st , stage ) . Right . Transheterozygous Rpp3018 . 2 / Rpp30PE ovarioles overexpressing germline specific nanosGal4 , UASRpp30GFP . Note follicular somatic with no Rpp30GFP expression ( arrowhead ) . Magnifications show Rpp30 localization , Rpp30 perinuclear foci ( arrows ) , Orb ( a specific oocyte marker ) . Scale bar , 10µm .

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E. Rpp30-dependent early oogenesis arrest is restored by expression of Rpp30GFP transgene in two different genetic backgrounds: Left. Homozygous Rpp3018.2 ovariole expressing ubiquitous ubiRpp30GFP (st, stage). Right. Transheterozygous Rpp3018.2/Rpp30PE ovarioles overexpressing germline specific nanosGal4,UASRpp30GFP. Note follicular somatic cells with no Rpp30GFP expression (arrowhead). Magnifications show nuclear Rpp30 localization, Rpp30 perinuclear foci (arrows), and Orb (a specific oocyte marker). Scale bar, 10µm.


F . flp / FRT clones mutant for Rpp3018 . 2specifically in germline ( star ) / or follicular ( arrows ) are detected by absence of GFP . Magnifications show mutant oocytes with Orb mislocalization next to karyosome ( dotted circle ) instead of being localized to posterior , as seen in stage 3 ( st . 3 ) wild - type clones . Scale bar , 10µm .

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F. flp/FRT clones mutant for Rpp3018.2specifically in the germline (star) and/or the follicular cells (arrows) are detected by the absence of GFP. Magnifications show mutant oocytes with Orb mislocalization next to the karyosome (dotted circle) instead of being localized to the posterior, as seen in stage 3 (st.3) wild-type clones. Scale bar, 10µm.


Figure 2 . Rpp30 is required for pre - tRNA processing . Whole fly extracts were used to study tRNA processing by Northern Blot . Top box : three different probes used are depicted : 5 ' 3 ' probes ( red green , respectively ) can detect full pre - tRNAs tRNAs intermediates , but will not detect mature tRNAs . An Internal Probe ( IP , blue ) corresponding to anti - codon region was used to detect non - mature mature tRNAs . genotypes used in this experiment are numbered at bottom ( 1 - 6 ) . Northern Blot panels : tRNA - his 5 ' probe ( top ) , tRNA - his 3 ' probe ( middle ) tRNA - his IP ( bottom ) . U6 probe was used as loading control .

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Figure 2. Rpp30 is required for pre-tRNA processing.Whole fly RNA extracts were used to study tRNA processing by Northern Blot. Top box : the three different probes used are depicted : 5' and 3' probes (red and green, respectively) can detect full pre-tRNAs and tRNAs intermediates, but will not detect mature tRNAs. An Internal Probe (IP, blue) corresponding to the anti-codon region was used to detect all non-mature and mature tRNAs. The genotypes used in this experiment are numbered at the bottom (1-6). Northern Blot panels: tRNA-his 5' probe (top), tRNA-his 3' probe (middle) and tRNA-his IP (bottom). U6 probe was used as loading control.


A . Rpp3018 . 2 homozygous early oogenesis arrest is partially rescued by p53 mutation . Scale bar , 100μm .

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A. Rpp3018.2 homozygous early oogenesis arrest is partially rescued by p53 mutation. Scale bar,100μm.


B . Early oogenesis arrest found in Rpp3018 . 2 homozygous ovaries is rescued by chk2 mutation ( Rpp3018 . 2 , mnkp6 Rpp3018 . 2 / Rpp30PE , mnkp6 ) . Scale bar , 100μm . Star : example of one rescued stage 9 egg chamber . Arrow : example of non - rescued egg chambers .

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B. Early oogenesis arrest found in Rpp3018.2 homozygous ovaries is rescued by chk2 mutation (Rpp3018.2 , mnkp6 and Rpp3018.2/ Rpp30PE, mnkp6). Scale bar, 100μm. Star: example of one rescued stage 9 egg chamber. Arrow: example of non-rescued egg chambers.


C . Germline clones mutant for Rpp3018 . 2 were immunostained for PCNA ( red ) . DAPI is in blue . Magnifications : PCNA signal in a control or mutant nurse . Scale bar , 10μm .

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C. Germline clones mutant for Rpp3018.2 were immunostained for PCNA (red). DAPI is in blue. Magnifications: PCNA signal in a control or mutant nurse cell. Scale bar, 10μm.


D . Germline clones mutant for Rpp3018 . 2 were immunostained for Brf ( pol - III ) ( red ) . DAPI is in blue . A Z - projection of Brf is shown . arrow points Brf in a mutant chamber . Scale bar , 10μm .

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D. Germline clones mutant for Rpp3018.2 were immunostained for Brf (pol-III) (red). DAPI is in blue. A Z-projection of Brf staining is shown. The arrow points Brf aggregates in a mutant chamber. Scale bar, 10μm.


E . Early oogenesis arrest found in Rpp3018 . 2 homozygous ovaries is partially rescued by claspin mutation ( Rpp3018 . 2 ; claspinEP / TM3 ) . Scale bar , 100μm .

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E. Early oogenesis arrest found in Rpp3018.2 homozygous ovaries is partially rescued by claspin mutation (Rpp3018.2; claspinEP/TM3). Scale bar, 100μm.


G . Top panel . Early oogenesis arrest found in Rpp30182 . / PEtransheterozygous ovaries is rescued by sqd - S - HAoverexpression . Scale bars : 100µm . Low panel . Rpp3018 . 2 / PE ; sqd - S - HA / sqd - S - HA ovaries were dissected stained for Orb ( green ) , HA ( squid , red ) DAPI ( blue ) . Scale bar , 10µm .

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G. Top panel. Early oogenesis arrest found in Rpp30182./PEtransheterozygous ovaries is rescued by sqd-S-HAoverexpression. Scale bars: 100µm. Low panel. Rpp3018.2/PE ; sqd-S-HA/sqd-S-HA ovaries were dissected and stained for Orb (green), HA (squid, red) and DAPI (blue). Scale bar, 10µm.


. D . Nuage specific markers ( Aub , Ago3 Mael ) are shown in stage 3 wild - type germline mutant clones for Rpp3018 . 2 . Scale bar , 10µm .

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. D. Nuage specific markers (Aub, Ago3 and Mael) are shown in stage 3 wild-type and germline mutant clones for Rpp3018.2. Scale bar, 10µm.


E - H . Double mutant ovaries for Rpp30 chk2 ( Rpp3018 . 2 , mnkp6 ) rescued specific general piRNA populations , nuage characteristic ping - pong signal . Scale bar , 10µm .

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E-H. Double mutant ovaries for Rpp30 and chk2 (Rpp3018.2, mnkp6) rescued the specific and general piRNA populations, nuage morphology and the characteristic ping-pong signal. Scale bar, 10µm.


D . Testes from control heterozygous , transheterozygous Rpp3018 . 2 / Rpp30PE or aubHN / QC42 were fixed stained for Stellate crystal formation . Z - projections are shown .

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D. Testes from control heterozygous, transheterozygous Rpp3018.2/Rpp30PE or aubHN/QC42 were fixed and stained for Stellate crystal formation. Z-projections are shown.


( A ) 3D time ‐ lapse microscopy of mitochondria undergoing fission in a dendritic arbor of a neuron . Neurons were transfected with Mito ‐ DsRed2 , pretreated with pan ‐ caspase inhibitor zVAD ‐ fmk methyl ester ( 100 μM ) , exposed to SNOC ( 200 μM ) . were 3D iso ‐ surface rendered . Frames depict representative time points of movie demonstrating mitochondrial fragmentation within 3 h of NO exposure ( upper panels ; scale bar , 15 μm ) closeup views ( lower panels ; scale bar , 3 μm ) . See Supplementary Video 1 .

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(A) 3D time‐lapse microscopy of mitochondria undergoing fission in a dendritic arbor of a neuron. Neurons were transfected with Mito‐DsRed2, pretreated with the pan‐caspase inhibitor zVAD‐fmk methyl ester (100 μM), and exposed to SNOC (200 μM). Images were 3D iso‐surface rendered. Frames depict representative time points of the movie demonstrating mitochondrial fragmentation within 3 h of NO exposure (upper panels; scale bar, 15 μm) and closeup views (lower panels; scale bar, 3 μm). See Supplementary Video 1.


( B ) Mitochondrial fragmentation within field of view shown in ( A ) results in increased mitochondrial numbers .

(B) Mitochondrial fragmentation within the field of view shown in (A) results in increased mitochondrial numbers.


( C ) NO induces dose ‐ dependent mitochondrial fission . Neurons expressing Mito ‐ DsRed2 were exposed to SNOC fixed at 60 min . fraction of neurons displaying fragmented mitochondria is shown as mean ± s . e . m .

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(C) NO induces dose‐dependent mitochondrial fission. Neurons expressing Mito‐DsRed2 were exposed to SNOC and fixed at 60 min. The fraction of neurons displaying fragmented mitochondria is shown as the mean±s.e.m.


( D ) Mito ‐ DsRed2 ‐ expressing corticalneurons were pretreated with 1 mM nitro ‐ L ‐ arginine ( 20 min ) exposed to 25 μM NMDA . fraction of neurons exhibiting fragmented mitochondria at 60 min after NMDA treatment is shown as mean ± s . e . m . of quintuplicate samples from a representative experiment ( † † † significance at P0 . 001 or n . s . † , not significant compared to control ; * * * significance at P0 . 001 as compared to NMDA treatment ) .

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(D) Mito‐DsRed2‐expressing corticalneurons were pretreated with 1 mM nitro‐L‐arginine (20 min) and exposed to 25 μM NMDA. The fraction of neurons exhibiting fragmented mitochondria at 60 min after NMDA treatment is shown as the mean±s.e.m. of quintuplicate samples from a representative experiment (†††significance at P0.001 or n.s.†, not significant compared to control; ***significance at P0.001 as compared to NMDA treatment).


Mitochondrial fission occurs before dendritic injury without AIF or cytochrome c release from mitochondria . Neurons were co ‐ transfected with vectors encoding ( A ) Mito ‐ DsRed2 ( B ) MyrPalm ‐ mCFP plus ( C ) AIF ‐ GFP effects of SNOC were recorded using 3D time ‐ lapse imaging . Projection zoomed in onto dendritic arbor of one neuron ( representative of n = 42 ) . Arrows indicate dendritic spines . inset shows soma at appropriate grayscale . apparent brightening of AIF ‐ GFP in nucleus at 5 h was due to shrinkage rather than translocation of AIF . Scale bars , 10 μm . See also Supplementary Video 2 .

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Mitochondrial fission occurs before dendritic injury and without AIF or cytochrome c release from mitochondria. Neurons were co‐transfected with vectors encoding (A) Mito‐DsRed2 and (B) MyrPalm‐mCFP plus (C) AIF‐GFP and the effects of SNOC were recorded using 3D time‐lapse imaging. Projection images zoomed in onto the dendritic arbor of one neuron (representative of n=42). Arrows indicate dendritic spines. The inset shows the soma at appropriate grayscale levels. The apparent brightening of AIF‐GFP fluorescence in the nucleus at 5 h was due to nuclear and cell body shrinkage rather than nuclear translocation of AIF. Scale bars, 10 μm. See also Supplementary Video 2.


( D ) 3D time ‐ lapse image reconstructions of mitochondria cytochrome c in a dendritic arbor at indicated time points following SNOC addition . Scale bar , 10 μm . Five mitochondria were selected motion tracked throughout imaging series .

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(D) 3D time‐lapse image reconstructions of mitochondria and cytochrome c in a dendritic arbor at indicated time points following SNOC addition. Scale bar, 10 μm. Five mitochondria were selected and motion tracked throughout the imaging series.


( E ) Traces show of mean GFP to DsRed2 emission from each mitochondrion . Each trace color corresponds to same colored mitochondrion . ( representative of n = 9 )

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(E) Traces show the ratio of mean GFP to DsRed2 emission intensity from each mitochondrion. Each trace color corresponds to the same colored mitochondrion. (representative of n=9)


( F - G ) Immunocytochemistry for anti ‐ AIF antibodies ( red ) anti ‐ NeuN antibodies ( green ) nuclei labeled with Hoechst 33342 dye ( blue ) after ( F ) aged SNOC or ( G ) SNOC ( 150 μM ; 4 h ) treatment . Volume rendered 3D reconstruction of confocal image stacks are shown above , a single confocal plane in grayscale below . Grid , 3 . 3 μm for ( F ) 1 . 9 μm for ( G ) , scale bars , 5 μm .

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(F-G) Immunocytochemistry for staining anti‐AIF antibodies (red) anti‐NeuN antibodies (green) and nuclei labeled with Hoechst 33342 dye (blue) after (F) aged SNOC or (G) SNOC (150 μM; 4 h) treatment. Volume rendered 3D reconstruction of confocal image stacks are shown above, and a single confocal plane in grayscale below. Grid, 3.3 μm for (F) and 1.9 μm for (G), scale bars, 5 μm.


( H ) of to somal AIF signal was measured as mean in center of nuclei divided by mean in , outlined by NeuN ( excluding nucleus ) . bar diagram summarizes mean ± s . e . m . of 282 262 neurons measured in n = 19 20 image stacks in three independent experiments for aged SNOC SNOC , respectively ( P ∼ 0 . 99 not significant by Student ' s t ‐ ) .

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(H) Ratio of nuclear to somal AIF signal was measured as mean fluorescence intensity in the center of nuclei divided by the mean fluorescence intensity in the cell body, outlined by the NeuN staining (excluding the nucleus). The bar diagram summarizes the mean±s.e.m. of 282 and 262 neurons measured in n=19 and 20 image stacks in three independent experiments for aged SNOC and SNOC, respectively (P∼0.99 not significant by Student's t‐test).


( I ) DEVDase activity . Purified cortical neurons were exposed to either 200 μM SNOC or 1 μM staurosporine , as positive control . rate of zDEVD ‐ AMC caspase substrate cleavage per minute was monitored expressed in arbitrary units . Data show mean ± s . e . m . for four independent experiments ( * significance at P0 . 01 by ANOVA ) .

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(I) DEVDase activity. Purified cortical neurons were exposed to either 200 μM SNOC or 1 μM staurosporine, as positive control. The rate of zDEVD‐AMC caspase substrate cleavage per minute was monitored and expressed in arbitrary fluorescence units. Data show mean±s.e.m. for four independent experiments (*significance at P0.01 by ANOVA).


( A ) Elongated mitochondrion ( arrow ) in neuronal process of unaffected hemisphere ( top ) . Small mitochondrial segments ( arrows ) are in close proximity to each other in a neuronal process of ischemic brain region ( bottom ) . are representative of three analyzed mice . Scale bars , 500 nm .

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(A) Elongated mitochondrion (arrow) in neuronal process of the unaffected hemisphere (top). Small mitochondrial segments (arrows) are in close proximity to each other in a neuronal process of the ischemic brain region (bottom). Images are representative of three analyzed mice. Scale bars, 500 nm.


( B ) Histogram comparing mitochondrial cross ‐ section of , control ( red ) ischemic ( black ) hemispheres . lower bins of histogram ( 2000 nm ) were compared with χ2 ‐ were not different . table shows presence of long mitochondrial cross ‐ sections ( > 2000 nm ) in control hemisphere their absence in ischemic hemisphere ( P0 . 01 ; Fisher ' s exact ) .

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(B) Histogram comparing mitochondrial cross‐section length of contralateral, control (red) and ischemic (black) hemispheres. The lower bins of the histogram (2000 nm) were compared with χ2‐test and were not different. The table shows the presence of long mitochondrial cross‐sections (>2000 nm) in the control hemisphere and their absence in the ischemic hemisphere (P0.01; Fisher's exact test).


( C ) Proximities of mitochondria comparing ( red ) ischemic ( black ) hemispheres . Bar diagram shows percents of mitochondrial proximities found to be smaller or larger than 400 nm . same data set is summarized as a contingency table on right , ( P0 . 001 ; two ‐ way Fisher ' s exact ) .

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(C) Proximities of mitochondria comparing the contralateral (red) and ischemic (black) hemispheres. Bar diagram shows percents of mitochondrial proximities found to be smaller or larger than 400 nm. The same data set is summarized as a contingency table on the right, (P0.001; two‐way Fisher's exact test).


Fission is associated with ultrastructural changes in mitochondria . EM tomography of mitochondria in cultured corticalneurons demonstrates injury associated with mitochondrial fission . Cortical cultures were exposed to aged SNOC control ( A - D ) or 200 μM SNOC ( E - L ) for 6 h in presence of 80 μM zVAD ‐ fmk methyl ester . A slice from each tilt series is shown ( A , E I ) , followed by orthogonal views of 3D reconstructions after segmentation of OMM ( blue ) IMM ( gray ) , with cristae ( various arbitrary colors ) ( B - D , F - H J - L ) . For clarity , 10 representative cristae are shown in ( B , D , F , G , J K ) , while cristae are displayed in ( C , H L ) . control mitochondrion ( A - D ) is elongated with a matrix , indicating no swelling , 34 cristae . NO ‐ exposed mitochondrion in ( E - H ) is dividing , lower mitochondrion of couplet shows severe damage , recognized by an outer rupture , a regionally confined inner blowout , slight matrix swelling cristae fragmentation . Each of daughter mitochondria is shorter rounder than controls . Unlike upper daughter , few of cristae extend throughout lower mitochondrion . Additionally , cristae are considerably smaller regionally confined . lower mitochondrion of couplet contains 82 cristae , while upper contains only 39 , indicating cristae fragmentation in former . NO ‐ exposed mitochondrial triplet in ( I - L ) displays further damage , including degradation , accumulation of an electron dense mass , greater cristae fragmentation , apparent mitochondrial fragmentation . cristae are smaller than controls , with 73 in top mitochondrion , 112 in middle , 47 in bottom . Scale bars , 400 nm . See Supplementary Video 3 , 4 5 of tomographic reconstruction .

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Fission is associated with ultrastructural changes in mitochondria. EM tomography of mitochondria in cultured corticalneurons demonstrates injury associated with mitochondrial fission. Cortical cultures were exposed to aged SNOC control (A-D) or 200 μM SNOC (E-L) for 6 h in the presence of 80 μM zVAD‐fmk methyl ester. A slice from each tilt series is shown (A, E and I), followed by orthogonal views of 3D reconstructions after segmentation of the OMM (blue) and IMM (gray), with cristae (various arbitrary colors) (B-D, F-H and J-L). For clarity, 10 representative cristae are shown in (B, D, F, G, J and K), while all cristae are displayed in (C, H and L). The control mitochondrion (A-D) is elongated with a dark matrix, indicating no swelling, and 34 cristae. The NO‐exposed mitochondrion in (E-H) is dividing, and the lower mitochondrion of the couplet shows severe damage, recognized by an outer membrane rupture, a regionally confined inner membrane blowout, slight matrix swelling and cristae fragmentation. Each of the daughter mitochondria is shorter and rounder than controls. Unlike the upper daughter, few of the cristae extend throughout the lower mitochondrion. Additionally, cristae are considerably smaller and regionally confined. The lower mitochondrion of the couplet contains 82 cristae, while the upper contains only 39, indicating cristae fragmentation in the former. The NO‐exposed mitochondrial triplet in (I-L) displays further damage, including membrane degradation, accumulation of an electron dense mass, greater cristae fragmentation, and apparent mitochondrial fragmentation. All cristae are smaller than controls, with 73 in the top mitochondrion, 112 in the middle, and 47 in the bottom. Scale bars, 400 nm. See Supplementary Video 3, 4 and 5 of tomographic reconstruction.


( A ) Projection image of two neurons expressing Mito ‐ DsRed2 ( red ) MyrPalm ‐ CFP ( blue ) . were high ‐ pass filtered z ‐ stacks were projected over maximum .

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(A) Projection image of two neurons expressing Mito‐DsRed2 (red) and MyrPalm‐CFP (blue). Images were high‐pass filtered and z‐stacks were projected over maximum intensity.


( B ) Changes in mitochondrial dynamics were evaluated as mean skeletal of mitochondria or ( C ) as mitochondrial numbers .

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(B) Changes in mitochondrial dynamics were evaluated as mean skeletal length of mitochondria or (C) as mitochondrial numbers.


( D ) Magnified representative frames of time ‐ lapse recording of within rectangle of ( A ) indicate mitochondrial at time 0 , 20 min 1 h . These time points are depicted in ( B ) ( C ) by red arrows . Data are representative of 26 neurons , which exhibited reversible mitochondrial fission from a total of 55 analyzed neurons ( six experiments ) . Scale bar , 10 μm . See Supplementary Video 6 .

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(D) Magnified representative frames of the time‐lapse recording of the area within the white rectangle of (A) indicate mitochondrial morphology at time 0, 20 min and 1 h. These time points are depicted in (B) and (C) by red arrows. Data are representative of 26 neurons, which exhibited reversible mitochondrial fission from a total of 55 analyzed neurons (six experiments). Scale bar, 10 μm. See Supplementary Video 6.


( E ) Autophagy in neurites at 6 h after SNOC exposure . Arrows indicate autophagosomes engulfing injured mitochondria next to an intact mitochondrion in left image . arrowhead in right panel indicates wrappers characteristic of autophagosomes . arrowheads in insets show double membranes of autophagosome those of mitochondria within autophagosome . Scale bars , 500 nm .

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(E) Autophagy in neurites at 6 h after SNOC exposure. Arrows indicate autophagosomes engulfing injured mitochondria next to an intact mitochondrion in the left image. The arrowhead in the right panel indicates membrane wrappers characteristic of autophagosomes. White arrowheads in the insets show the double membranes of the autophagosome and those of mitochondria within the autophagosome. Scale bars, 500 nm.


( A ) Mitochondrial fission is linked to a drop in ATP . Purified corticalneurons were exposed to increasing SNOC concentrations or mitochondrial inhibitors ( 2 μM rotenone plus 2 μg / ml oligomycin ; Rot / Olig ) . ATP concentrations are shown as mean ± s . e . m . normalized to plating density of neurons ( n = 6 )

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(A) Mitochondrial fission is linked to a drop in ATP. Purified corticalneurons were exposed to increasing SNOC concentrations or mitochondrial inhibitors (2 μM rotenone plus 2 μg/ml oligomycin; Rot/Olig). ATP concentrations are shown as the mean±s.e.m. normalized to the plating density of neurons (n=6)


( B ) [ ATP ] / [ ADP ] ratios in purified corticalneurons exposed to increasing SNOC concentrations ( n = 4 ) .

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(B) [ATP]/[ADP] ratios in purified corticalneurons exposed to increasing SNOC concentrations (n=4).


( C ) bar graph depicts ethidine density in arbitrary units of aged fresh SNOC treated neurons at 2 . 5 h .

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(C) The bar graph depicts ethidine fluorescence density in arbitrary units of aged and fresh SNOC treated neurons at 2.5 h.


( D ) Cortical neurons expressing Mito ‐ GFP ( green ) were exposed to aged or fresh 200 μM SNOC solution . After 2 . 5 h cultures were loaded with hydroethidine . are representative of more that 20 neurons analyzed ( in D ) for each condition from at least three independent experiments . Scale bar , 10 μm .

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(D) Cortical neurons expressing Mito‐GFP (green) were exposed to aged or fresh 200 μM SNOC solution. After 2.5 h cultures were loaded with hydroethidine. Images are representative of more that 20 neurons analyzed (in D) for each condition from at least three independent experiments. Scale bar, 10 μm.


( E ) Reduced glutathione ( GSH ) partially blocks mitochondrial fission . Neurons were transfected with Mito pretreated for 2 h with 2 mM GSH monoethyl ester , washed once then exposed to 35 μM SNOC . Mitochondrial fission was scored after 1 h . Data are means ± s . e . m . of quintuplicate samples from one representative experiment of a total of three ( † † † significance at P0 . 001 or n . s . , † not significant compared to control ; * significance at P0 . 05 ) .

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(E) Reduced glutathione (GSH) partially blocks mitochondrial fission. Neurons were transfected with Mito pretreated for 2 h with 2 mM GSH monoethyl ester, washed once and then exposed to 35 μM SNOC. Mitochondrial fission was scored after 1 h. Data are means±s.e.m. of quintuplicate samples from one representative experiment of a total of three (†††significance at P0.001 or n.s., †not significant compared to control; *significance at P0.05).


( A ) Representative of mitochondrial before after SNOC ( 175 μM ; 7 h ) or rotenone ( 30 nM ; 2 h ) treatments a comparison of effects of Aβ25 - 35 versus Aβ35 - 25 ( 10 μM , 6 h ) , as indicated . Scale bar , 20 μm .

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(A) Representative fluorescence micrographs of mitochondrial morphology before and after SNOC (175 μM; 7 h) or rotenone (30 nM; 2 h) treatments and a comparison of the effects of Aβ25-35 versus Aβ35-25(10 μM, 6 h), as indicated. Scale bar, 20 μm.


( B ) of mitochondrial fission at 18 h ( † † † † significance at P0 . 05 0 . 001 as compared to control pcDNA3 transfection ; * , * * * * * significance at P0 . 05 , 0 . 01 0 . 001 , respectively , compared to SNOC treated , pcDNA3 transfected neurons ; n = 3 independent experiments ) . Dying neurons were recognized by their shrunken condensed nuclei after Hoechst 33342 .

(B) Percentage of mitochondrial fission and cell death at 18 h († and ††† significance at P0.05 and 0.001 as compared to control pcDNA3 transfection; *,** and *** significance at P0.05 , 0.01 and 0.001, respectively, compared to SNOC treated, pcDNA3 transfected neurons; n=3 independent experiments). Dying neurons were recognized by their shrunken and condensed nuclei after Hoechst 33342 staining.


( C ) Enforced Drp1 or Fis1 expression ( 60 h ) evokes fission neuronal ( * , * * * * * significance at P0 . 05 , 0 . 01 0 . 001 , respectively , compared to pcDNA3 transfected neurons ; n = 3 independent experiments ) .

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(C) Enforced Drp1 or Fis1 expression (60 h) evokes fission and neuronal cell death (*,** and *** significance at P0.05, 0.01 and 0.001, respectively, compared to pcDNA3 transfected neurons; n=3 independent experiments).


( D ) Rotenone induces dose ‐ dependent mitochondrial fission . fraction of neurons displaying fissioned mitochondria is shown as mean ± s . e . m . ( * significance at P0 . 05 , n = 4 ) .

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(D) Rotenone induces dose‐dependent mitochondrial fission. The fraction of neurons displaying fissioned mitochondria is shown as the mean ±s.e.m. (*significance at P0.05, n=4).


( E ) Mfn1 or Drp1K38A inhibits 100 nM rotenone ‐ induced mitochondrial fission ( 4 h ) ( 48 h ) . Data indicate means ± s . e . m . of triplicate measurements ( † † † † † significance at P0 . 01 0 . 001 as compared to untreated control ; * * * significance at P0 . 001 as compared to rotenone treatment with pcDNA3 transfection ; n = 3 ) .

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(E) Mfn1 or Drp1K38A inhibits 100 nM rotenone‐induced mitochondrial fission (4 h) and cell death (48 h). Data indicate means±s.e.m. of triplicate measurements (†† and ††† significance at P0.01 and 0.001 as compared to untreated control; ***significance at P0.001 as compared to rotenone treatment with pcDNA3 transfection; n=3).


( F ) Mitochondrial fission by Aβ35 - 25 or Aβ25 - 35 exposure . fraction of neurons exhibiting fragmented mitochondria is shown as mean ± s . e . m . ( † † significance at P0 . 01 compared to pcDNA3 transfected , Aβ35 − 25 treated control ; * * * significance at P0 . 05 0 . 001 , respectively , compared to pcDNA3 transfected , Aβ25 - 35 treated neurons ; n = 3 ) .

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(F) Mitochondrial fission by Aβ35-25 or Aβ25-35 exposure. The fraction of neurons exhibiting fragmented mitochondria is shown as the mean±s.e.m. (††significance at P0.01 compared to pcDNA3 transfected, Aβ35−25 treated control; * and ** significance at P0.05 and 0.001, respectively, compared to pcDNA3 transfected, Aβ25-35 treated neurons; n=3).


A , B Co - immunoprecipitation ( IP ) of EBNA2 ( A ) PLK1 ( B ) using EBNA2 - , PLK1 - specific antibodies , or isotype - matched control antibodies performed on total lysates ( L ) of lymphoblastoid long - term B cultures ( LCLs ) infected with recombinant EBV 2089 , which is on EBV B95 -

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A, B Co-immunoprecipitation (IP) of EBNA2 (A) and PLK1 (B) using EBNA2-, PLK1-specific antibodies, or isotype-matched control antibodies performed on total cell lysates (L) of lymphoblastoid long-term B cell cultures (LCLs) infected with recombinant EBV strain 2089, which is based on EBV strain B95-8


GFP - tagged EBNA2 fragments to co - precipitate endogenous PLK1 . Total lysates immunoprecipitates ( IP ) were analyzed by Western blotting ( WB ) .

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GFP-tagged EBNA2 fragments to co-precipitate endogenous PLK1. Total protein lysates and immunoprecipitates (IP) were analyzed by Western blotting (WB).


( H ) Co - immunoprecipitation of transfected EBNA2 wt or S379A endogenous PLK1 .

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(H) Co-immunoprecipitation of transfected EBNA2 wt or S379A and endogenous PLK1.


( D ) EBNA2 candidate phosphorylation mutants were expressed in DG75 B tested for PLK1 binding by co - immunoprecipitations followed by Western blotting .

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(D) EBNA2 candidate phosphorylation mutants were expressed in DG75 B cells and tested for PLK1 binding by co-immunoprecipitations followed by Western blotting.


( F ) GST EBNA2 fragment 246 - 487 wt mutant S457A / T465V were treated with recombinant active PLK1 ( + ) in presence of [ γ - 32P ] ATP in vitro or left untreated ( - ) . CRS , an artificial PLK1 substrate was used as a positive control .

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(F) GST EBNA2 fragment 246-487 wt and mutant S457A/T465V were treated with recombinant active PLK1 (+) in the presence of [γ-32P] ATP in vitro or left untreated (-). CRS, an artificial PLK1 test substrate was used as a positive control.


( H ) HA - tagged EBNA2 candidate phosphorylation mutants were expressed in DG75 B tested for activation of an EBNA2 / CBF1 responsive promoter reporter luciferase plasmid . Activation of reporter gene is shown as relative response normalized to Renilla luciferase activity shown relative to wt activity .

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(H) HA-tagged EBNA2 candidate phosphorylation mutants were expressed in DG75 B cells and tested for activation of an EBNA2/CBF1 responsive promoter reporter luciferase plasmid. Activation of the reporter gene is shown as relative response ratio normalized to Renilla luciferase activity and shown relative to wt activity.