E Expression of + 1 + 2 ( CGG ) 100 NL - 3xF plasmid reporters with without an AUG inserted 5 ' to CGG repeat , with without DDX3X knockdown . Black asterisks refer to comparisons between siDDX3X - siEGFP - treated ; orange asterisks refer to comparisons between siDDX3X - treated expressing either + 1 or + 2 ( CGG ) 100 NL - 3xF those expressing respective AUG - driven variant ( two - way ANOVA with Tukey ' s multiple comparisons ; n = 11 - 12 / condition ) . Data Information : For panels , ns = non - significant , * * * P ≤ 0 . 001 , * * * * P ≤ 0 . 0001 for specified statistical . panels depict data as means ± SD , unless indicated otherwise ( compiled from ≥ 3 replicates ) .

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E Expression of +1 and +2 (CGG)100 NL-3xF plasmid reporters with and without an AUG inserted 5' to the CGG repeat, with and without DDX3X knockdown. Black asterisks refer to comparisons between siDDX3X- and siEGFP-treated cells; orange asterisks refer to comparisons between siDDX3X-treated cells expressing either +1 or +2 (CGG)100 NL-3xF and those expressing the respective AUG-driven variant (two-way ANOVA with Tukey's multiple comparisons test; n=11-12/condition). Data Information: For all panels, ns=non-significant, *** P≤0.001, **** P≤0.0001 for the specified statistical test. All panels depict data as means ± SD, unless indicated otherwise (compiled from ≥3 replicates).


F Expression of NL - 3xF plasmids with initiator AUG codons mutated to near - AUG codons , with without DDX3X knockdown ( two - way ANOVA with Dunnett ' s multiple comparisons ; n = 18 - 24 / condition ) . Black asterisks refer to comparisons between siEGFP - treated siDDX3X - treated ; orange asterisks refer to comparisons between siDDX3X - treated expressing AUG - NL - 3xF those expressing a different reporter ; asterisks refer to comparisons between siDDX3X - treated expressing + 1 ( CGG ) 100 NL - 3xF those expressing a different reporter . Data Information : For panels , ns = non - significant , * * * P ≤ 0 . 001 , * * * * P ≤ 0 . 0001 for specified statistical . panels depict data as means ± SD , unless indicated otherwise ( compiled from ≥ 3 replicates ) .

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F Expression of NL-3xF plasmids with initiator AUG codons mutated to near-AUG codons, with and without DDX3X knockdown (two-way ANOVA with Dunnett's multiple comparisons test; n=18-24/condition). Black asterisks refer to comparisons between siEGFP-treated and siDDX3X-treated cells; orange asterisks refer to comparisons between siDDX3X-treated cells expressing AUG-NL-3xF and those expressing a different reporter; white asterisks refer to comparisons between siDDX3X-treated cells expressing +1 (CGG)100 NL-3xF and those expressing a different reporter. Data Information: For all panels, ns=non-significant, *** P≤0.001, **** P≤0.0001 for the specified statistical test. All panels depict data as means ± SD, unless indicated otherwise (compiled from ≥3 replicates).


G Expression of in vitro transcribed near - AUG reporter RNAs in in vitro translation extracts , collected from HeLa treated with siRNAs against EGFP or DDX3X . ( n = 4 / group ) .

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G Expression of in vitro transcribed near-AUG reporter RNAs in in vitro translation extracts, collected from HeLa cells treated with siRNAs against EGFP or DDX3X. (n=4/group).


A Representative of GMR - GAL4 ; ( CGG ) 90 - EGFP fly eyes expressing manipulations of eIF4B eIF4H . ( B ) Quantitation of GMR - GAL4 , ( CGG ) 90 - EGFP eye with eIF4B / H manipulations ( Mann - Whitney U with Bonferroni corrections for multiple comparisons ; n = 26 - 55 / genotype ) . Data Information : For panels , ns = non - significant , * P ≤ 0 . 05 , * * P ≤ 0 . 01 , * * * * P ≤ 0 . 0001 for specified statistical . panels present data as means ± SD ( compiled from ≥ 3 replicates ) .

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A Representative photographs of GMR-GAL4; (CGG)90-EGFP fly eyes expressing manipulations of eIF4B and eIF4H. (B) Quantitation of GMR-GAL4, (CGG)90-EGFP eye phenotypes with eIF4B/H manipulations (Mann-Whitney U test with Bonferroni corrections for multiple comparisons; n=26-55/genotype). Data Information: For all panels, ns=non-significant, * P≤0.05, ** P≤0.01, **** P≤0.0001 for the specified statistical test. All panels present data as means ± SD (compiled from ≥3 replicates).


C , D Expression of plasmid - AUG - NL + 1 ( CGG ) 100 NL - 3xF reporters ( C ) , or co - transfected AUG - FF reporters ( D ) , following knockdown of EIF4B or EIF4H . Black asterisks refer to comparisons between siEGFP - siEIF4B / H - treated ; pink blue asterisks refer to comparisons between siEIF4B - ( pink ) or siEIF4H - ( blue ) treated expressing AUG - NL - 3xF those expressing + 1 ( CGG ) 100 ( two - way ANOVA with Tukey ' s multiple comparisons , n = 9 / condition ) .

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C, D Expression of plasmid-based AUG-NL and +1 (CGG)100 NL-3xF reporters (C), or co-transfected AUG-FF reporters (D), following knockdown of EIF4B or EIF4H. Black asterisks refer to comparisons between siEGFP- and siEIF4B/H-treated cells; pink and blue asterisks refer to comparisons between siEIF4B- (pink) or siEIF4H- (blue) treated cells expressing AUG-NL-3xF and those expressing +1 (CGG)100 (two-way ANOVA with Tukey's multiple comparisons test, n=9/condition).


E Expression of plasmid - AUG - NL - 3xF ( CGG ) 100 + 1 NL - 3xF reporters with without over - expression of EIF4B , EIF4H , or both ( two - way ANOVA with Dunnett ' s multiple comparisons ; n = 20 / condition ) . Asterisks refer to comparisons between over - expressing either EGFP or EIF4B , EIF4H , or EIF4B EIF4H expressing same reporter . Data Information : For panels , ns = non - significant , * P ≤ 0 . 05 , * * P ≤ 0 . 01 , * * * * P ≤ 0 . 0001 for specified statistical . panels present data as means ± SD ( compiled from ≥ 3 replicates ) .

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E Expression of plasmid-based AUG-NL-3xF and (CGG)100 +1 NL-3xF reporters with and without over-expression of EIF4B, EIF4H, or both (two-way ANOVA with Dunnett's multiple comparisons test; n=20/condition). Asterisks refer to comparisons between cells over-expressing either EGFP or EIF4B, EIF4H, or EIF4B and EIF4H and expressing the same reporter. Data Information: For all panels, ns=non-significant, * P≤0.05, ** P≤0.01, **** P≤0.0001 for the specified statistical test. All panels present data as means ± SD (compiled from ≥3 replicates).


A Expression of plasmid - NL - 3xF reporters in HEK293 with without over - expression of EIF1 ( two - way ANOVA with Sidak ' s multiple comparisons ; n = 9 - 12 / condition ) . Black asterisks refer to comparisons between Empty Vector - transfected EIF1 - transfected ; green asterisks refer to comparisons between EIF1 - transfected expressing AUG - NL - 3xF those expressing a different reporter . For panels , * * * P ≤ 0 . 001 , * * * * P ≤ 0 . 0001 for specified statistical . Bars represent mean ± ( compiled from ≥ 3 replicates ) .

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A Expression of plasmid-based NL-3xF reporters in HEK293 cells with and without over-expression of EIF1 (two-way ANOVA with Sidak's multiple comparisons test; n=9-12/condition). Black asterisks refer to comparisons between Empty Vector-transfected and EIF1-transfected cells; green asterisks refer to comparisons between EIF1-transfected cells expressing AUG-NL-3xF and those expressing a different reporter. For all panels, *** P≤0.001, **** P≤0.0001 for the specified statistical test. Bars represent mean ± SEM (compiled from ≥3 replicates).


B Expression of plasmid - NL - 3xF reporters in HEK293 with without over - expression of EIF5 ( two - way ANOVA with Sidak ' s multiple comparisons ; n = 9 - 12 / condition ) . Black asterisks refer to comparisons between Empty Vector - transfected EIF5 - transfected ; pink asterisks refer to comparisons between EIF5 - transfected expressing AUG - NL - 3xF those expressing a different reporter . For panels , * * * P ≤ 0 . 001 , * * * * P ≤ 0 . 0001 for specified statistical . Bars represent mean ± ( compiled from ≥ 3 replicates ) .

B Expression of plasmid-based NL-3xF reporters in HEK293 cells with and without over-expression of EIF5 (two-way ANOVA with Sidak's multiple comparisons test; n=9-12/condition). Black asterisks refer to comparisons between Empty Vector-transfected and EIF5-transfected cells; pink asterisks refer to comparisons between EIF5-transfected cells expressing AUG-NL-3xF and those expressing a different reporter. For all panels, *** P≤0.001, **** P≤0.0001 for the specified statistical test. Bars represent mean ± SEM (compiled from ≥3 replicates).


A Sample collected by automated longitudinal microscopy , demonstrating automated determination of .

A Sample micrographs collected by automated longitudinal fluorescence microscopy, demonstrating the automated determination of cell death.


B Anti - DDX3X western blot of B35 transfected with either of two independent anti - DDX3X LNAs or a control LNA .

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B Anti-DDX3X western blot of B35 cells transfected with either of two independent anti-DDX3X LNAs or a control LNA.


C Expression of EGFP in rat neurons transfected with ( CGG ) 100 ( + 1 ) EGFP either anti - DDX3X LNAs or a control ( one - way ANOVA with Tukey ' s multiple comparisons ; n = 2408 - 5689 / condition ) . graphs depict pooled data , normalized first within replicate . Data Information : For panels , * * * * P ≤ 0 . 0001 for specified statistical ( compiled from ≥ 3 replicates ) .

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C Expression of EGFP in primary rat neurons transfected with (CGG)100 (+1) EGFP and either anti-DDX3X LNAs or a control (one-way ANOVA with Tukey's multiple comparisons test; n=2408-5689 cells/condition). All graphs depict pooled data, normalized first within the replicate. Data Information: For all panels, **** P≤0.0001 for the specified statistical test (compiled from ≥3 replicates).


D , E Transfection of anti - DDX3X LNA # 1 ( D ) or # 2 ( E ) reduced cumulative risk of in ( CGG ) 100 ( + 1 ) EGFP - expressing neurons ( Cox proportional hazard ; n = 2408 - 3676 / condition ) . Data Information : For panels , * * * * P ≤ 0 . 0001 for specified statistical ( compiled from ≥ 3 replicates ) .

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D, E Transfection of anti-DDX3X LNA #1 (D) or #2 (E) reduced the cumulative risk of death in (CGG)100 (+1) EGFP-expressing neurons (Cox proportional hazard analysis; n=2408-3676 cells/condition). Data Information: For all panels, **** P≤0.0001 for the specified statistical test (compiled from ≥3 replicates).


A Immunofluorescence of MMS22L foci formation at etoposide ( ETP ) - induced DSBs during S , M G1 phases of cycle in HeLa . Scale bar : 5 μm . B of a representative experiment from ( A ) shows number of MMS22L foci per nucleus during indicated cycle phases ( n = 2 ; nnuclei ≥ 60 ) .

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A Immunofluorescence images of MMS22L foci formation at etoposide (ETP)-induced DSBs during S, M and G1 phases of the cell cycle in HeLa cells. Scale bar: 5 μm.B Quantification of a representative experiment from (A) shows number of MMS22L foci per nucleus during indicated cell cycle phases (n = 2; nnuclei ≥ 60).


C Immunofluorescence of CPT - induced MMS22L , RPA2 , BRCA2 RAD51 foci in U2OS . Scale bar : 5 μm . D of a representative experiment ( n = 3 ) from ( C ) shows of γH2AX , RPA2 , BRCA2 RAD51 foci co - localizing with MMS22L foci ( nnuclei = 30 ; nuclei with ≥ 25 MMS22L foci were analyzed ) .

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C Immunofluorescence images of CPT-induced MMS22L, RPA2, BRCA2 and RAD51 foci in U2OS cells. Scale bar: 5 μm.D Quantification of a representative experiment (n = 3) from (C) shows percentage of γH2AX, RPA2, BRCA2 and RAD51 foci co-localizing with MMS22L foci (nnuclei = 30; nuclei with ≥ 25 MMS22L foci were analyzed).


A Electrophoretic mobility shift monitoring MMS22L - TONSL binding to ssDNA without RPA with RPA - coated ssDNA . B of data as in ( A ) shows MMS22L - TONSL binding to ssDNA with or without RPA , n = 2 ; error bars , .

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A Electrophoretic mobility shift assay monitoring MMS22L-TONSL binding to ssDNA without RPA and with RPA-coated ssDNA.B Quantification of data as in (A) shows percent MMS22L-TONSL binding to ssDNA with or without RPA, n = 2; error bars, SEM.


C Schematic ( left ) western blots ( right ) of streptavidin NiNTA pulldowns of his - tagged MMS22L - TONSL , RPA biotinylated ssDNA .

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C Schematic (left) and western blots (right) of streptavidin and NiNTA pulldowns of his-tagged MMS22L-TONSL, RPA and biotinylated ssDNA.


D Yeast two - hybrid shows effect of deletion of 384 - 522 of MMS22L on interaction with RPA . interaction between indicated MMS22L variants fused to LexA - binding domain RPA1 fused to GAL4 activating domain ( GAD ) is assessed by survival on media containing 3 - aminotriazole ( 3 - AT ) in absence of histidine by β - galactosidase activity ( X - Gal ) assays .

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D Yeast two-hybrid analysis shows the effect of deletion of the amino acids 384-522 of MMS22L on the interaction with RPA. The interaction between indicated MMS22L variants fused to LexA DNA-binding domain and RPA1 fused to GAL4 activating domain (GAD) is assessed by survival on media containing 3-aminotriazole (3-AT) in the absence of histidine and by β-galactosidase activity (X-Gal) assays.


E of MMS22L foci formed in U2OS stably expressing wild - type ( WT ) or Δ384 - 522 variants of siRNA - resistant HSS - MMS22L , depleted of endogenous MMS22L by RNAi treated with 50 nM CPT for 18 hours . Scale bar : 5 μm .

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E Images of MMS22L foci formed in U2OS cells stably expressing wild-type (WT) or Δ384-522 variants of siRNA-resistant HSS-MMS22L, depleted of endogenous MMS22L by RNAi and treated with 50 nM CPT for 18 hours. Scale bar: 5 μm.


( A - E ) iPOND assays in Hela . Experiment schematic ( top ) resulting immunoblots ( bottom ) are shown in ( A , B , D ) quantifications are shown in ( C , E ) with antibodies used for immunoblots indicated throughout . A Abundance of MMS22L - TONSL at active HU - stalled replication forks , fully replicated .

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(A-E) iPOND assays in Hela cells. Experiment schematic (top) and resulting immunoblots (bottom) are shown in (A, B, D) and quantifications are shown in (C, E) with antibodies used for immunoblots indicated throughout.A Abundance of MMS22L-TONSL at active and HU-stalled replication forks, and fully replicated DNA.


( A - E ) iPOND assays in Hela . Experiment schematic ( top ) resulting immunoblots ( bottom ) are shown in ( A , B , D ) quantifications are shown in ( C , E ) with antibodies used for immunoblots indicated throughout . B , C MMS22L dynamics at stalled replication forks . shows average relative abundance of indicated detected in immunoblots , n = 3 except of time - point 5 h , n = 1 ; error bars , .

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(A-E) iPOND assays in Hela cells. Experiment schematic (top) and resulting immunoblots (bottom) are shown in (A, B, D) and quantifications are shown in (C, E) with antibodies used for immunoblots indicated throughout.B, C MMS22L dynamics at stalled replication forks. Quantification shows average relative abundance of indicated proteins detected in immunoblots, n = 3 except of time-point 5 h, n = 1; error bars, SEM.


( A - E ) iPOND assays in Hela . Experiment schematic ( top ) resulting immunoblots ( bottom ) are shown in ( A , B , D ) quantifications are shown in ( C , E ) with antibodies used for immunoblots indicated throughout . D , E Effect of MMS22L depletion on RAD51 at stalled replication forks . shows average relative RAD51 abundance , n = 2 ; error bars , .

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(A-E) iPOND assays in Hela cells. Experiment schematic (top) and resulting immunoblots (bottom) are shown in (A, B, D) and quantifications are shown in (C, E) with antibodies used for immunoblots indicated throughout.D, E Effect of MMS22L depletion on RAD51 levels at stalled replication forks. Quantification shows average relative RAD51 abundance, n = 2; error bars, SEM.


A Representative immunofluorescence of foci induced by short CPT treatment ( 50 nM , 1 h ) in U2OS treated with indicated siRNAs showing effect of MMS22L depletion on RAD51 foci . Scale bar : 5 μm . B Boxplot of representative experiment in ( A ) ( n = 3 ; nnuclei ≥ 78 ) . Boxes indicate 25 - 75 percentile whiskers 10 - 90 percentile ; horizontal lines mark medians . Nuclei with ≥ 25 γH2AX foci ( marker of replication stress sites ) were analyzed . Statistical : Mann - Whitney U ; * * * P ≤ 0 . 0001 .

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A Representative immunofluorescence images of foci induced by short CPT treatment (50 nM, 1 h) in U2OS cells treated with indicated siRNAs showing the effect of MMS22L depletion on the RAD51 foci. Scale bar: 5 μm.B Boxplot quantification of representative experiment in (A) (n = 3; nnuclei ≥ 78). Boxes indicate the 25-75 percentile and whiskers the 10-90 percentile; horizontal lines mark
the medians. Nuclei with ≥ 25 γH2AX foci (marker of replication stress sites) were analyzed. Statistical analysis: Mann-Whitney U test; *** P ≤ 0.0001.


C , D Electron of a representative reversed fork ( C ) a fork with extended ssDNA region at junction ( D ) ( arrow ) . P , parental duplex ; R , regressed arm . E Frequency of reversed replication forks detected by EM . Similar results were obtained in one independent experiment ; n = 2 . F Distribution of ssDNA at replication forks isolated from U2OS transfected with indicated siRNAs treated with 25 nM CPT for 1h . In brackets , total number of analyzed molecules is given . Statistical according to Mann - Whitney U ; * * P ≤ 0 . 01 , n = 2 .

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C, D Electron micrographs of a representative reversed fork (C) and a fork with extended ssDNA region at the junction (D) (arrow). P, parental duplex; R, regressed arm.E Frequency of reversed replication forks detected by EM. Similar results were obtained in one independent experiment; n = 2.F Distribution of ssDNA length at replication forks isolated from U2OS cells transfected with the indicated siRNAs and treated with 25 nM CPT for 1h. In brackets, the total number of analyzed molecules is given. Statistical analysis according to Mann-Whitney U test; ** P ≤ 0.01, n = 2.


A Amylose pulldown of MBP ( control ) or MBP - tagged MMS22L - TONSL RAD51 . Eluate were detected by silver . MT : MBP - MMS22L - GST - TONSL . B Quantitative of RAD51 binding to MMS22L - TONSL such as shown in Figure EV6A . Averages shown , n = 2 ; error bars , .

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A Amylose pulldown of MBP (control) or MBP-tagged MMS22L-TONSL and RAD51. Eluate proteins were detected by silver staining. MT: MBP-MMS22L-GST-TONSL.B Quantitative analysis of RAD51 binding to MMS22L-TONSL such as shown in Figure EV6A. Averages shown, n = 2; error bars, SEM.


C , D Yeast two - hybrid of interactions between LexA binding domain - tagged MMS22L indicated fused to GAL4 activating domain ( GAD ) assessed by survival on media containing 3 - AT in absence of histidine by X - Gal assays .

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C, D Yeast two-hybrid analysis of interactions between LexA DNA binding domain-tagged MMS22L and indicated proteins fused to GAL4 activating domain (GAD) assessed by survival on media containing 3-AT in the absence of histidine and by X-Gal assays.


E , F Streptavidinpulldowns of indicated recombinant biotinylated ssDNA . in input eluates were detected by western blotting .

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E, F Streptavidinpulldowns of indicated recombinant proteins and biotinylated ssDNA. The proteins in the input and eluates were detected by western blotting.


G Streptavidinpulldown of HSS - tagged MMS22L variants stably integrated overexpressed in U2OS . Input co - precipitated were detected by immunoblotting .

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G Streptavidinpulldown of HSS-tagged MMS22L variants stably integrated and overexpressed in U2OS cells. Input and co-precipitated proteins were detected by immunoblotting.


H Representative immunofluorescence of CPT - induced foci in stable U2OS lines depleted of endogenous MMS22L overexpressing indicated siRNA - resistant variants of HSS - MMS22L treated with 50 nM CPT for 1 hour . Scale bar : 5 μm .

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H Representative immunofluorescence images of CPT-induced foci in stable U2OS cell lines depleted of endogenous MMS22L and overexpressing indicated siRNA-resistant variants of HSS-MMS22L treated with 50 nM CPT for 1 hour. Scale bar: 5 μm.


I - K of effect of indicated MMS22L mutations on RAD51 foci in U2OS treated with 50 nM CPT for 1 h ( I ; n = 3 ) , 50 nM CPT for 18 h ( J ; n = 3 ) or 5 µM ETP for 1 h followed by 3 hours incubation without drug ( K ; n = 3 ) . Graphs represent averaged median values ; error bars indicate . Statistical according to one - way ANOVA with Bonferroni post - ; * * * P ≤ 0 . 001 ; * * P ≤ 0 . 01 ; ns , not significant . Representative immunofluorescence are shown in ( H ) , Figures EV6G EV6H , respectively .

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I-K Quantification of the effect of indicated MMS22L mutations on RAD51 foci in U2OS cells treated with 50 nM CPT for 1 h (I; n = 3), 50 nM CPT for 18 h (J; n = 3) or 5 µM ETP for 1 h followed by 3 hours incubation without the drug (K; n = 3). Graphs represent averaged median values; error bars indicate SEM. Statistical analysis according to one-way ANOVA with Bonferroni post-test; *** P ≤ 0.001; ** P ≤ 0. 01; ns, not significant. Representative immunofluorescence images are shown in (H), Figures EV6G and EV6H, respectively.


B - D Strand exchange with ssDNA RAD51 , with ( C ) or without ( B ) addition of MMS22L - TONSL ( 75 nM ) . D shows averages , n = 2 ; error bars , .

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B-D Strand exchange assay with ssDNA and RAD51, and with (C) or without (B) the addition of MMS22L-TONSL (75 nM).D Quantification shows averages, n = 2; error bars, SEM.


E Averages of quantified strand exchange assays with ssDNA , RAD51 ( 270 nM ) varying concentrations of MMS22L - TONSL or MMS22L . n = 2 ; error bars , .

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E Averages of quantified strand exchange assays with ssDNA, RAD51 (270 nM) and varying concentrations of MMS22L-TONSL or MMS22L. n = 2; error bars, SEM.


F of electrophoretic mobility shift assays with dsDNA or ssDNA , RAD51 MMS22L - TONSL or MMS22L . Averages shown , n = 2 ; error bars , .

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F Quantification of electrophoretic mobility shift assays with dsDNA or ssDNA, RAD51 and MMS22L-TONSL or MMS22L. Averages shown, n = 2; error bars, SEM.


H A representative immunoblot showing effect of increasing amounts of dsDNA on RAD51 binding to ssDNA ( 1 nM ) .

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H A representative immunoblot showing the effect of increasing amounts of dsDNA on RAD51 binding to ssDNA (1 nM).


I A representative immunoblot showing effects of MMS22L - TONSL MMS22L titration into reactions containing RAD51 , ssDNA ( 1 nM ) dsDNA ( 10 nM ) , as indicated .

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I A representative immunoblot showing effects of MMS22L-TONSL and MMS22L titration into reactions containing RAD51, ssDNA (1 nM) and dsDNA (10 nM), as indicated.


( A ) After 4 h of starvation in 1 % K ‐ acetate medium in presence of PMSF , autophagic vesicles can be visualized in vacuoles of wild ‐ type pep4prb1 using Normaski optics . In aut2Δ aut7Δ no autophagic vesicles can be seen . Bar represents 20 μm .

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(A) After 4 h of starvation in 1% K‐acetate medium in the presence of PMSF, autophagic vesicles can be visualized in vacuoles of wild‐type and pep4prb1 cells using Normaski optics. In aut2Δ and aut7Δ cells no autophagic vesicles can be seen. Bar represents 20 μm.


( B ) Total breakdown of starved on nitrogen ‐ free medium is significantly reduced in aut2Δ aut7Δ compared with wild ‐ type . pep4Δ ( pra1Δ ) are almost completely impaired in starvation ‐ induced vacuolar breakdown . were ‐ labeled with [ 35S ] methionine chased on non ‐ radioactive starvation medium . Aliquots were taken at indicated times acid soluble small peptides generated by proteolysis were determined according to Straub et al . ( 1997 ) .

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(B) Total protein breakdown of cells starved on nitrogen‐free medium is significantly reduced in aut2Δ and aut7Δ cells compared with wild‐type cells. pep4Δ (pra1Δ) cells are almost completely impaired in starvation‐induced vacuolar protein breakdown. Cells were pulse‐labeled with [35S]methionine and chased on non‐radioactive starvation medium. Aliquots were taken at the indicated times and acid soluble small peptides generated by proteolysis were determined according to Straub et al. (1997).


( C ) Survival during starvation . Like pep4Δ ( pra1Δ ) , which are almost completely impaired in vacuolar proteolysis , aut2Δ aut7Δ exhibit a significantly reduced survival rate during starvation on nitrogen ‐ free medium as compared with wild ‐ type . Aliquots of incubated in 1 % K ‐ acetate were plated growing colonies were determined according to Straub et al . ( 1997 ) .

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(C) Survival during starvation. Like pep4Δ (pra1Δ) cells, which are almost completely impaired in vacuolar proteolysis, aut2Δ and aut7Δ cells exhibit a significantly reduced survival rate during starvation on nitrogen‐free medium as compared with wild‐type cells. Aliquots of cells incubated in 1% K‐acetate were plated and growing colonies were determined according to Straub et al. (1997).


Maturation of proaminopeptidase I is impaired in aut2 ‐ 1 ( A ) , aut2Δ ( B ) aut7Δ ( C ) . Further details are outlined in text . Crude extracts were prepared from starved 4 h on 1 % K ‐ acetate .

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Maturation of proaminopeptidase I is impaired in aut2‐1 (A), aut2Δ (B) and aut7Δ (C) cells. Further details are outlined in the text. Crude extracts were prepared from cells starved 4 h on 1% K‐acetate.


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( A ) Detection of - interactions using two ‐ hybrid system . Intense indicates an interaction between Aut2p Aut7p , Aut2p Tub1p , Aut2p Tub2p . AUT7 , TUB1 TUB2 are cloned in ‐ frame with GAL4 ‐ activator domain . AUT2 AUT7 were fused to GAL4 ‐ binding domain ( see Materials methods ) .

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(A) Detection of protein-protein interactions using the two‐hybrid system. Intense staining indicates an interaction between Aut2p and Aut7p, Aut2p and Tub1p, and Aut2p and Tub2p. AUT7, TUB1 and TUB2 are cloned in‐frame with the GAL4‐activator domain. AUT2 and AUT7 were fused to the GAL4‐binding domain (see Materials and methods).


( B ) Affinity chromatography confirms - interaction between Aut2p Aut7p . A fusion of Aut2p with 26 kDa glutathione ‐ S ‐ transferase ( GST ) domain from Schistosoma japonicum ( see Materials methods ) was bound to glutathione - Sepharose 4B . This GST - Aut2p column was incubated with a crude extract from aut7Δ cen Met25 : : AUT7 - GFP . non ‐ bound supernatant ( S ) fraction ( lane 1 ) was taken away . Gel beads were successively washed with NaCl solutions of increasing concentrations to remove non ‐ specifically bound . After final 500 mM NaCl wash ( W , lane 2 ) , bound were eluted with Laemmli buffer ( E , lane 3 ) . Samples were analyzed in immunoblots with monoclonal antibodies directed against GFP . Using Aut2p - GST , a clear band corresponding to Aut7p - GFP is seen in eluate fraction ( lane 3 , arrowhead ) . As a control , GST alone was attached to column no band was detectable ( lane 6 ) .

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

(B) Affinity chromatography confirms protein-protein interaction between Aut2p and Aut7p. A fusion protein of Aut2p with the 26 kDa glutathione‐S‐transferase (GST) domain from Schistosoma japonicum (see Materials and methods) was bound to glutathione-Sepharose 4B. This GST-Aut2p column was incubated with a crude extract from aut7Δ cen Met25::AUT7-GFP cells. The non‐bound supernatant (S) fraction (lane 1) was taken away. Gel beads were successively washed with NaCl solutions of increasing concentrations to remove non‐specifically bound proteins. After the final 500 mM NaCl wash (W, lane 2), bound proteins were eluted with Laemmli buffer (E, lane 3). Samples were analyzed in immunoblots with monoclonal antibodies directed against GFP. Using Aut2p-GST, a clear band corresponding to Aut7p-GFP is seen in the eluate fraction (lane 3, arrowhead). As a control, GST alone was attached to the column and no band was detectable (lane 6).


( C ) As described in ( B ) , bovine tubulin binds to GST - Aut2p glutathione - Sepharose 4B ( lane 12 ) , but not to GST glutathione - Sepharose 4B ( lane 9 ) . with Coomassie is shown in lanes 1 - 6 ; in lanes 7 - 12 tubulin was detected using monoclonal antibodies ( Boehringer Mannheim ) . Further details are given in Materials methods .

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(C) As described in (B), bovine tubulin binds to GST-Aut2p glutathione-Sepharose 4B (lane 12), but not to GST glutathione-Sepharose 4B (lane 9). Protein staining with Coomassie is shown in lanes 1-6; in lanes 7-12 tubulin was detected using monoclonal antibodies (Boehringer Mannheim). Further details are given in Materials and methods.


( D ) Similar to ( C ) , bovine tubulin does not bind to GST - Aut7p glutathione - Sepharose 4B ( lane 12 ) to GST glutathione - Sepharose 4B ( lane 9 ) . Lanes 1 - 6 , with Coomassie ; lanes 7 - 12 probed with antibodies directed against tubulin .

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(D) Similar to (C), bovine tubulin does not bind to GST-Aut7p glutathione-Sepharose 4B (lane 12) and to GST glutathione-Sepharose 4B (lane 9). Lanes 1-6, protein staining with Coomassie; lanes 7-12 probed with antibodies directed against tubulin.


Benomyl sensitivity of wild ‐ type overexpressing AUT2 AUT7 , of aut2Δ aut7Δ is not altered . were grown to stationary phase dilution series with decreasing densites were dropped on CM plates without uracil containing 15 30 μg / ml benomyl . Plates were incubated at 30 ° C .

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Benomyl sensitivity of wild‐type cells overexpressing AUT2 and AUT7, and of aut2Δ and aut7Δ cells is not altered. Cells were grown to stationary phase and dilution series with decreasing cell densites were dropped on CM plates without uracil containing 15 and 30 μg/ml benomyl. Plates were incubated at 30°C.


( A ) Autophagosome ‐ like vesicles ( arrowhead ) are accumulating in cytoplasm of aut2Δ starved for 4 h on 1 % K ‐ acetate in presence of PMSF . Bar represents 100 nm .

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(A) Autophagosome‐like vesicles (arrowhead) are accumulating in the cytoplasm of aut2Δ cells starved for 4 h on 1% K‐acetate in the presence of PMSF. Bar represents 100 nm.


( B ) Electron unravels accumulation of double ‐ layered autophagosome ‐ like vesicles in cytoplasm of aut7Δ starved for 3 h in nitrogen ‐ free medium in presence of PMSF . Arrowheads indicate clearly distinguishable two layers . Bar represents 100 nm .

O O O O O O O O O O O O O O O O O O O O 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) Electron micrographs unravels the accumulation of double membrane‐layered autophagosome‐like vesicles in the cytoplasm of aut7Δ cells starved for 3 h in nitrogen‐free medium in the presence of PMSF. Arrowheads indicate the clearly distinguishable two membrane layers. Bar represents 100 nm.


( C D ) Cytosol containing autophagosome ‐ like vesicles accumulating in cytosol of aut2Δ ( D ) can be isolated with an additional 100 000 g centrifugation step yielding a P100 pellet fraction . In wild ‐ type ( C ) , no P100 is detectable . Fatty acid synthase ( Fas ) was used as a . Further details are given in text .

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(C and D) Cytosol containing autophagosome‐like vesicles accumulating in the cytosol of aut2Δ cells (D) can be isolated with an additional 100 000 g centrifugation step yielding a P100 pellet fraction. In wild‐type cells (C), no P100 is detectable. Fatty acid synthase (Fas) was used as a cytosolic protein. Further details are given in the text.


( D , E ) HTM - mediated of MPM - 2 ( D ) H3S10P ( E ) per nucleus in U2OS treated with DMSO as a control or 50 μM P22077 ( USP7i ) for 4 hrs . Nuclei were stained with DAPI ( blue ) . Scale bar , 30 μm . is shown to right of representative for each . ( * * * p < 0 . 05 ; t - ) . Grey lines indicate mean values .

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(D, E) HTM-mediated quantification of MPM-2 (D) and H3S10P (E) levels per individual nucleus in U2OS cells treated with DMSO as a control or 50 μM P22077 (USP7i) for 4 hrs. Nuclei were stained with DAPI (blue). Scale bar, 30 μm. The quantification is shown to the right of representative images for each analysis. (*** p<0.05; t-test). Grey lines indicate mean values.


( A ) Western Blot showing of USP7 , MPM - 2 , CDK1Y15P CDK1 in whole extracts of RPE treated with 25 μM P22077 for 2 - 4 hrs ( USP7i ) , an asterisk indicates a non - specific band .

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(A) Western Blot showing the levels of USP7, MPM-2, CDK1Y15P and CDK1 in whole cell extracts of RPE cells treated with 25 μM P22077 for 2-4 hrs (USP7i), an asterisk indicates a non-specific band.


( B ) Immunofluorescence of CCNB1 ( red ) CCNB1S126P ( green ) in RPE in control conditions ( Control ) after treatment with 25 μM P22077 for 2 hrs ( USP7i ) . Nuclei were stained with DAPI ( blue ) . Scale bar , 30 μm .

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(B) Immunofluorescence of CCNB1 (red) and CCNB1S126P (green) in RPE cells in control conditions (Control) and after treatment with 25 μM P22077 for 2 hrs (USP7i). Nuclei were stained with DAPI (blue). Scale bar, 30 μm.


( B ) Western blot showing of MPM - 2 , pAKT , AKT , pPRC1 , pKAP , KAP , pMAPKAPK - 2 , pSTAT3 , STAT3 β - Actin in whole extracts of RPE treated with DMSO ( Control ) , 0 . 5 μM Okadaic acid for 2 hrs , 25 μM P22077 for 4 hrs or 25 μM BAY 11 - 7082 for 2 hrs . Ponceau is shown as loading control .

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(B) Western blot showing the levels of MPM-2, pAKT, AKT, pPRC1, pKAP, KAP, pMAPKAPK-2, pSTAT3, STAT3 and β-Actin in whole cell extracts of RPE cells treated with DMSO (Control), 0.5 μM Okadaic acid for 2 hrs, 25 μM P22077 for 4 hrs or 25 μM BAY 11-7082 for 2 hrs. Ponceau staining is shown as loading control.


( D ) Immunofluorescence of PP2A - A ( green ) in U2OS treated with DMSO ( CONTROL ) , 50 μM P22077 ( USP7i ) for 4 hrs or 0 . 5 μM Okadaic acid ( OA ) for 1 hr 30 min . Nuclei were stained with DAPI ( blue ) . Scale bar , 10 μm .

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(D) Immunofluorescence of PP2A-A (green) in U2OS cells treated with DMSO (CONTROL), 50 μM P22077 (USP7i) for 4 hrs or 0.5 μM Okadaic acid (OA) for 1 hr 30 min. Nuclei were stained with DAPI (blue). Scale bar, 10 μm.


( E ) Western blot showing of MPM - 2 , pAKT , AKT , pPRC1 , pMAPKAPK - 2 , p4e - BP1 , 4e - BP1 β - Actin , in whole extracts of RPE treated with DMSO ( Control ) , 25 μM P22077 ( USP7i ) 10 μM of PP2A activator DT - 061 for 2 - 4 hrs . Ponceau is shown as loading control .

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(E) Western blot showing the levels of MPM-2, pAKT, AKT, pPRC1, pMAPKAPK-2, p4e-BP1, 4e-BP1 and β-Actin, in whole cell extracts of RPE cells treated with DMSO (Control), 25 μM P22077 (USP7i) and 10 μM of the PP2A activator DT-061 for 2-4 hrs. Ponceau staining is shown as loading control.


( F ) Western blot showing of MPM - 2 , phosphor - CDK substrate motif [ ( K / H ) pSP ] β - Actin in whole extracts of RPE treated with DMSO ( Control ) , 0 . 5 μM Okadaic acid ( OA ) for 2 hrs , 10 nM Calyculin A for 2 hrs , 25 μM P22077 for 4 hrs or 25 μM BAY 11 - 7082 for 2 hrs . Calyculin A was added to this experiment to illustrate gain in CDK activity induced by an additional PP2A inhibitor . Ponceau is shown as loading control .

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(F) Western blot showing the levels of MPM-2, phosphor-CDK substrate motif [(K/H)pSP] and β-Actin in whole cell extracts of RPE cells treated with DMSO (Control), 0.5 μM Okadaic acid (OA) for 2 hrs, 10 nM Calyculin A for 2 hrs, 25 μM P22077 for 4 hrs or 25 μM BAY 11-7082 for 2 hrs. Calyculin A was added to this experiment to illustrate the gain in CDK activity induced by an additional PP2A inhibitor. Ponceau staining is shown as loading control.


( E ) Western blot showing of USP7 γH2AX in whole extracts of HCT - 116 treated with DMSO ( Control ) , 50 μM P22077 for 2 - 4 hrs ( USP7i ) , 10 μM RO3306 for 4 hrs ( CDK1i ) or 50 μM P22077 + 10 μM RO3306 for 2 - 4 hrs ( USP7i + CDK1i ) . Ponceau is shown as loading control .

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(E) Western blot showing the levels of USP7 and γH2AX in whole cell extracts of HCT-116 cells treated with DMSO (Control), 50 μM P22077 for 2-4 hrs (USP7i), 10 μM RO3306 for 4 hrs (CDK1i) or 50 μM P22077 + 10 μM RO3306 for 2-4 hrs (USP7i + CDK1i). Ponceau staining is shown as loading control.


( B ) of apoptotic induction was measured by flow cytometry in RPE after treatment with DMSO as a control , 25 μM P22077 ( USP7i ) , 10 μM RO3306 ( CDK1i ) or 10 μM DT - 061 ( PP2Aa ) , their combinations for indicated times . content was followed with Hoechst , mitochondrial potential was measured by TMRE . of apoptotic ( TMRE negative ) is indicated for each condition . This experiment was repeated 3 times . ( * p < 0 . 05 , * * p < 0 . 01 , t - ) . Bars represent mean values ± .

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(B) Quantification of apoptotic induction was measured by flow cytometry in RPE cells after treatment with DMSO as a control, 25 μM P22077 (USP7i), 10 μM RO3306 (CDK1i) or 10 μM DT-061 (PP2Aa), and their combinations for the indicated times. DNA content was followed with Hoechst, and the mitochondrial membrane potential was measured by TMRE. The percentage of apoptotic cells (TMRE negative) is indicated for each condition. This experiment was repeated 3 times. (* p<0.05, ** p<0.01, t-test). Bars represent mean values ± SEM.


( C ) GLO viability in treated with 3 independent USP7 inhibitors ( P22077 , BAY 11 - 7082 or HBX 19818 ) alone or in combination with a 24hr treatment of 10 μM RO3306 ( CDK1i ) . This experiment was repeated 3 times . Data represent mean values ± . ( * * * p < 0 . 05 , ANOVA ) .

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(C) GLO viability assay in cells treated with 3 independent USP7 inhibitors (P22077, BAY 11-7082 or HBX 19818) alone or in combination with a 24hr treatment of 10 μM RO3306 (CDK1i). This experiment was repeated 3 times. Data represent mean values ± SEM. (*** p<0.05, ANOVA).


( a ) 30 150 days post fertilization ( dpf ) zebrafish ventricles were isolated relative determined ( n = 3 ) .

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

(a) 30 and 150 days post fertilization (dpf) zebrafish ventricles were isolated and relative protein levels determined (n = 3).


( c ) qPCR of mRNA of glycolytic enzyme genes in 5 dpci remote wound border cardiac tissue ( n = 2 - 3 technical replicates using pooled cDNA from 10 ventricles for each condition ( n = 2 for pkmb pdk4 , n = 3 for other genes ) ) .

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

(c) qPCR analysis of mRNA levels of glycolytic enzyme genes in 5 dpci remote and wound border cardiac tissue (n = 2-3 technical replicates using pooled cDNA from 10 ventricles for each condition (n = 2 for pkmb and pdk4, and n = 3 for the other genes)).


d , Immunostaining of heart sections for PCNA MEF2 in 5 dpci treated with PBS , 2 - DG or DCA ; magnified view of in boxes shown below ; dashed lines mark wound border ; arrowheads point to PCNA + CMs ; of PCNA + CMs in border zone shown on right ( n = 4 - 5 ventricles ) .

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

d, Immunostaining of heart sections for PCNA and MEF2 in 5 dpci animals treated with PBS, 2-DG or DCA; magnified view of area in white boxes shown below; white dashed lines mark the wound border; arrowheads point to PCNA+ CMs; percentage of PCNA+ CMs in the border zone shown on the right (n = 4-5 ventricles).


e ) Immunostaining of heart sections for MEF2 N2 . 261 MEF2 in 5 dpci treated with PBS , 2 - DG or DCA ; magnified view of in boxes shown below ; dashed lines mark wound border ; arrowheads point to N2 . 261 + CMs ; of N2 . 261 + CMs in border zone shown on right ( n = 4 - 5 ventricles ) .

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

e) Immunostaining of heart sections for MEF2 N2.261 and MEF2 in 5 dpci animals treated with PBS, 2-DG or DCA; magnified view of area in white boxes shown below; white dashed lines mark the wound border; arrowheads point to N2.261+ CMs; percentage of N2.261+ CMs in the border zone shown on the right (n = 4-5 ventricles).


( a Immunostaining of heart sections for PCNA MEF2 in 5 dpci pkma2 + / - ; pkmb + / - pkma2 - / - ; pkmb + / - ; magnified view of in boxes shown below ; arrowheads point to PCNA + CMs ; dashed lines mark wound border ; of PCNA + CMs in border zone shown on right ( n = 4 - 6 ventricles of each genotype ) .

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

(a Immunostaining of heart sections for PCNA and MEF2 in 5 dpci pkma2+/-; pkmb+/- and pkma2-/-; pkmb+/- animals; magnified view of area in white boxes shown below; arrowheads point to PCNA+ CMs; white dashed lines mark the wound border; percentage of PCNA+ CMs in the border zone shown on the right (n = 4-6 ventricles of each genotype).


Immunostaining of heart sections for N2 . 261 MEF2 in 5 dpci pkma2 + / - ; pkmb + / - pkma2 - / - ; pkmb + / - ; magnified view of in boxes shown below ; arrowheads point to N2 . 261 + CMs ; dashed lines mark wound border ; of N2 . 261 + in border zone shown on right ( n = 4 - 6 ventricles of each genotype ) .

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

Immunostaining of heart sections for N2.261 and MEF2 in 5 dpci pkma2+/-; pkmb+/- and pkma2-/-; pkmb+/- animals; magnified view of area in white boxes shown below; arrowheads point to N2.261+ CMs; white dashed lines mark the wound border; percentage of N2.261+ in the border zone shown on the right (n = 4-6 ventricles of each genotype).


Immunostaining of heart sections for PCNA MEF2 in 5 dpci ppargc1a + / + ppargc1a - / - ; magnified view of in boxes shown below ; arrowheads point to PCNA + CMs ; dashed lines mark wound border ; of PCNA + CMs in border zone shown on right ( n = 4 - 6 ventricles of each genotype ) .

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

Immunostaining of heart sections for PCNA and MEF2 in 5 dpci ppargc1a+/+ and ppargc1a-/- animals; magnified view of area in white boxes shown below; arrowheads point to PCNA+ CMs; white dashed lines mark the wound border; percentage of PCNA+ CMs in the border zone shown on the right (n = 4-6 ventricles of each genotype).


d ) Immunostaining of heart sections for N2 . 261 MEF2 in 5 dpci ppargc1a + / + ppargc1a - / - ; magnified view of in boxes shown below ; arrowheads point to N2 . 261 + CMs ; dashed lines mark wound border ; of N2 . 261 + CMs in border zone shown on right ( n = 4 - 6 ventricles of each genotype ) .

O O O O O O O O O O B-MEASURED_VAR O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Immunostaining of heart sections for N2.261 and MEF2 in 5 dpci ppargc1a+/+ and ppargc1a-/- animals; magnified view of area in white boxes shown below; arrowheads point to N2.261+ CMs; white dashed lines mark the wound border; percentage of N2.261+ CMs in the border zone shown on the right (n = 4-6 ventricles of each genotype).


( e AFOG of heart sections from 60 dpci pkma2 + / - ; pkmb + / - pkma2 - / - ; pkmb + / - ; black dashed lines outline scar ; scar measured on right ( n = 3 - 5 ventricles of each genotype ) .

O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O B-CONTROLLED_VAR O O O O O O O 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 AFOG staining of heart sections from 60 dpci pkma2+/-; pkmb+/- and pkma2-/-; pkmb+/- animals; black dashed lines outline the scar area; scar area measured on the right (n = 3-5 ventricles of each genotype).


f ) AFOG of heart sections from 45 dpci ppargc1a + / + ppargc1a - / - ; black dashed lines outline scar ; scar measured on right ( n = 3 - 5 ventricles of each genotype ) .

O O O O O 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

f) AFOG staining of heart sections from 45 dpci ppargc1a+/+ and ppargc1a-/- animals; black dashed lines outline the scar area; scar area measured on the right (n = 3-5 ventricles of each genotype).


Immunostaining of heart sections for PCNA MEF2 in 5 dpci ( Tg ( hsp70l : LOXP - STOP - LOXP - pdha1aSTA - T2A - mCherry ) Tg ( hsp70l : LOXP - STOP - LOXP - pdk3b - T2A - mCherry ) alone ( control ) or in combination with Tg ( myl7 : Cre - ERT2 ) , after tamoxifen heat shock treatments ) ; magnified view of in boxes shown below ; arrowheads point to PCNA + CMs ; dashed lines mark wound border ; of PCNA + CMs in border zone shown on right ( n = 4 - 5 ventricles of each genotype ) .

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 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 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

Immunostaining of heart sections for PCNA and MEF2 in 5 dpci animals (Tg(hsp70l:LOXP-STOP-LOXP-pdha1aSTA-T2A-mCherry) and Tg(hsp70l:LOXP-STOP-LOXP-pdk3b-T2A-mCherry) alone (control) or in combination with Tg(myl7:Cre-ERT2), all after tamoxifen and heat shock treatments); magnified view of area in white boxes shown below; arrowheads point to PCNA+ CMs; white dashed lines mark the wound border; percentage of PCNA+ CMs in the border zone shown on the right (n = 4-5 ventricles of each genotype).


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 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-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


Immunostaining of heart sections for N2 . 261 MEF2 in 5 dpci ( Tg ( hsp70l : LOXP - STOP - LOXP - pdha1aSTA - T2A - mCherry ) Tg ( hsp70l : LOXP - STOP - LOXP - pdk3b - T2A - mCherry ) alone ( control ) or in combination with Tg ( myl7 : Cre - ERT2 ) , after tamoxifen heat shock treatments ) ; magnified view of in boxes shown below ; arrowheads point to N2 . 261 + CMs ; dashed lines mark wound border ; of N2 . 261 + CMs in border zone shown on right ( n = 4 - 5 ventricles of each genotype ) .

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

Immunostaining of heart sections for N2.261 and MEF2 in 5 dpci animals (Tg(hsp70l:LOXP-STOP-LOXP-pdha1aSTA-T2A-mCherry) and Tg(hsp70l:LOXP-STOP-LOXP-pdk3b-T2A-mCherry) alone (control) or in combination with Tg(myl7:Cre-ERT2), all after tamoxifen and heat shock treatments); magnified view of area in white boxes shown below; arrowheads point to N2.261+ CMs; white dashed lines mark the wound border; percentage of N2.261+ CMs in the border zone shown on the right (n = 4-5 ventricles of each genotype).


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 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


( f AFOG of heart sections from 60 dpci ( Tg ( hsp70l : LOXP - STOP - LOXP - pdha1aSTA - T2A - mCherry ) alone ( control ) or in combination with Tg ( myl7 : Cre - ERT2 ) , after tamoxifen heat shock treatments ) ; black dashed lines outline scar ; scar measured on right ( n = 4 - 5 ventricles of each genotype ) .

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

(f AFOG staining of heart sections from 60 dpci animals (Tg(hsp70l:LOXP-STOP-LOXP-pdha1aSTA-T2A-mCherry) alone (control) or in combination with Tg(myl7:Cre-ERT2), all after tamoxifen and heat shock treatments); black dashed lines outline the scar area; scar area measured on the right (n = 4-5 ventricles of each genotype).


g ) AFOG of heart sections from 60 dpci Tg ( hsp70l : LOXP - STOP - LOXP - pdk3b - T2A - mCherry ) alone ( control ) or in combination with Tg ( myl7 : Cre - ERT2 ) , after tamoxifen heat shock treatments ) ; black dashed lines outline scar ; scar measured on right ( n = 4 - 5 ventricles of each genotype ) .

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

g) AFOG staining of heart sections from 60 dpci animals and Tg(hsp70l:LOXP-STOP-LOXP-pdk3b-T2A-mCherry) alone (control) or in combination with Tg(myl7:Cre-ERT2), all after tamoxifen and heat shock treatments); black dashed lines outline the scar area; scar area measured on the right (n = 4-5 ventricles of each genotype).


( a , b ) for Ki67 , CTNI ( DAPI ) in control , PDHA1STA OE or PDK3 OE RNCMs cultured in growth ( a ) or non - growth ( b ) medium ; arrowheads point to Ki67 + RNCMs ; of Ki67 + RNCMs shown on right ( n = 4 biological replicates for each condition ) .

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

(a, b) Staining for Ki67, CTNI and DNA (DAPI) in control, PDHA1STA OE or PDK3 OE RNCMs cultured in growth (a) or non-growth (b) medium; arrowheads point to Ki67+ RNCMs; percentage of Ki67+ RNCMs shown on the right (n = 4 biological replicates for each condition).


( c ) for CTNI ( DAPI ) in scratch using control , PDHA1STA OE or PDK3 OE RNCMs immediately or at 3 days after generating scratch ; magnified view of in boxes shown below ; arrowheads point to protrusions of RNCMs ; of CM protrusions shown on right ( n = 4 biological replicates for each condition ) .

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

(c) Staining for CTNI and DNA (DAPI) in scratch assay using control, PDHA1STA OE or PDK3 OE RNCMs immediately or at 3 days after generating the scratch; magnified view of area in white boxes shown below; arrowheads point to membrane protrusions of RNCMs; quantification of CM membrane protrusions shown on the right (n = 4 biological replicates for each condition).


( d ) Reactome over - representation of differentially regulated genes in PDK3 OE RNCMs compared to control .

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

(d) Reactome over-representation analysis of differentially regulated genes in PDK3 OE RNCMs compared to control.


( e Heat map of gene expression for key regulators of cycle in PDK3 OE vs control RNCMs .

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

(e Heat map of gene expression for key regulators of cell cycle in PDK3 OE vs control RNCMs.


f ) Heat map of gene expression for key regulators of replication in PDK3 OE vs control RNCMs .

f) Heat map of gene expression for key regulators of DNA replication in PDK3 OE vs control RNCMs.


( a ) H3R expression in cultured ratcortical neurons at DIV7 after OGD / R injury ( 2 h OGD 24 h reperfusion ) shown by western blot ( n = 6 per condition ; * * P0 . 01 versus control with Student ' s t - ) . Full - blots are shown in Supplementary Fig . 9 .

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

(a) H3R expression in cultured ratcortical neurons at DIV7 after OGD/R injury (2 h OGD and 24 h reperfusion) shown by western blot (n=6 per condition; **P0.01 versus control with Student's t-test). Full-size blots are shown in Supplementary Fig. 9.


( b ) When H3R antagonist thioperamide ( THIO , 10 − 6 mol l − 1 at reperfusion ) agonist immepip ( IMME , 10 − 6 mol l − 1 at reperfusion ) were administered , viability was tested in cultured neurons by 3 - ( 4 , 5 - dimethylthiazol - 2 - yl ) - 2 , 5 - diphenyltetrazolium bromide ( MTT ) after OGD / R ( n = 7 per condition ; * * * P0 . 001 with of variances ( ANOVAs ) followed by Bonferroni / Dunn post hoc ) .

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 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

(b) When the H3R antagonist thioperamide (THIO, 10−6 mol l−1 at reperfusion) and the agonist immepip (IMME, 10−6 mol l−1 at reperfusion) were administered, viability was tested in cultured neurons by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay after OGD/R (n=7 per condition; ***P0.001 with analysis of variances (ANOVAs) followed by the Bonferroni/Dunn post hoc test).


( c ) 2 , 3 , 5 - triphenyltetrazolium chloride - stained brain sections showing infarct in WT H3R − / − mice receiving saline , H3R antagonists A331440 , clobenpropit ( CLOB ) thioperamide , ( THIO ; 5 mg kg − 1 , i . p . , immediately at reperfusion 6 h later ) , H3R agonist immepip ( IMME , 1 μg , i . c . v . , at reperfusion ) after I / R ( 1 h MCAO followed by 24 h reperfusion ; scale bar , 5 mm ) .

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

(c) 2,3,5-triphenyltetrazolium chloride-stained brain sections showing the infarct area in WT and H3R−/− mice receiving saline, the H3R antagonists A331440, clobenpropit (CLOB) and thioperamide, (THIO; 5 mg kg−1, i.p., immediately at reperfusion and 6 h later), and the H3R agonist immepip (IMME, 1 μg, i.c.v., at reperfusion) after I/R (1 h MCAO followed by 24 h reperfusion; scale bar, 5 mm).


bar graphs show infarct volume ( d ) neurological score ( e ) ( n = 6 - 8 mice per condition , * P0 . 05 , * * P0 . 01 , * * * P0 . 001 versus WT saline group with ANOVAs followed by Bonferroni / Dunn post hoc ) . Data are presented as mean ± s . e . m .

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

The bar graphs show infarct volume (d) and neurological score (e) (n=6-8 mice per condition, *P0.05, **P0.01, ***P0.001 versus WT saline group with ANOVAs followed by the Bonferroni/Dunn post hoc test). Data are presented as mean±s.e.m.


( a ) TUNEL of apoptotic in penumbral brain slices in vivo ( scale bar , 100 μm ) when H3R was inhibited with thioperamide ( scale bar , 50 μm ) after tMCAO . TUNEL - positive are green , are stained with 4 ' , 6 - diamidino - 2 - phenylindole ( blue ) .

O 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

(a) TUNEL staining of apoptotic cells in penumbral brain slices in vivo (scale bar, 100 μm) when H3R was inhibited with thioperamide (scale bar, 50 μm) after tMCAO. TUNEL-positive cells are green, and all cells are stained with 4',6-diamidino-2-phenylindole (blue).


( b , d ) H3R was inhibited by thioperamide , caspase - 3 expression is shown by western blot in WT H3R − / − mice after tMCAO ( b ) ( n = 6 - 7 per condition ; * * P0 . 01 , * * * P0 . 001 with of variances ( ANOVAs ) followed by Bonferroni / Dunn post hoc )

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

(b,d) H3R was inhibited by thioperamide, and cleaved caspase-3 protein expression is shown by western blot in WT and H3R−/−mice after tMCAO (b) (n=6-7 per condition; **P0.01, ***P0.001 with analysis of variances (ANOVAs) followed by Bonferroni/Dunn post hoc test)


( c ) TUNEL of apoptotic in cultured neurons when H3R was inhibited with thioperamide ( scale bar , 50 μm ) after OGD / R . TUNEL - positive are green , are stained with 4 ' , 6 - diamidino - 2 - phenylindole ( blue ) .

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

(c) TUNEL staining of apoptotic cells in cultured neurons when H3R was inhibited with thioperamide (scale bar, 50 μm) after OGD/R. TUNEL-positive cells are green, and all cells are stained with 4',6-diamidino-2-phenylindole (blue).


( b , d ) H3R was inhibited by thioperamide , caspase - 3 expression is shown by western blot in WT H3R − / − mice in cultured neurons subjected to OGD / R ( d ) ( n = 8 per condition ; * P0 . 05 , * * * P0 . 001 with ANOVAs followed by Bonferroni / Dunn post hoc ) . Data are presented as mean ± s . e . m . Full - blots are shown in Supplementary Fig . 10 .

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

(b,d) H3R was inhibited by thioperamide, and cleaved caspase-3 protein expression is shown by western blot in WT and H3R−/−mice in cultured neurons subjected to OGD/R (d) (n=8 per condition; *P0.05, ***P0.001 with ANOVAs followed by the Bonferroni/Dunn post hoc test). Data are presented as mean±s.e.m. Full-size blots are shown in Supplementary Fig. 10.


( a ) 2 , 3 , 5 - Triphenyltetrazolium chloride ( TTC ) - stained brain sections from WT HDC − / − mice showing infarct in those receiving saline , H3R antagonist thioperamide ( THIO ) , H1R antagonist pyrilamine ( PYRI ; 10 mg kg − 1 , i . p . , at reperfusion ) , H2R antagonist cimetidine ( CIME ; 10 mg kg − 1 , i . p . , at reperfusion ) α - FMH ( 50 mg kg − 1 , i . p . , 2 h before ischemia ) after 24 h reperfusion ( n = 6 per condition ; scale bar , 5 mm ) .

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

(a) 2,3,5-Triphenyltetrazolium chloride (TTC)-stained brain sections from WT and HDC−/− mice showing the infarct area in those receiving saline, the H3R antagonist thioperamide (THIO), the H1R antagonist pyrilamine (PYRI; 10 mg kg−1, i.p., at reperfusion), the H2R antagonist cimetidine (CIME; 10 mg kg−1, i.p., at reperfusion) and α-FMH (50 mg kg−1, i.p., 2 h before ischemia) after 24 h reperfusion (n=6 per condition; scale bar, 5 mm).


( b ) Infarct volumes ( c ) neurological deficit scores ( * P0 . 05 , * * P0 . 01 , * * * P0 . 001 with of variances followed by Bonferroni / Dunn post hoc ) . Data are presented as mean ± s . e . m .

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

(b) Infarct volumes and (c) neurological deficit scores (*P0.05, **P0.01, ***P0.001 with analysis of variances followed by the Bonferroni/Dunn post hoc test). Data are presented as mean±s.e.m.


( a ) Akt / GSK - 3β / mTOR / P70S6K phosphorylation assessed by western blots after thioperamide administration OGD / R . LiCl ( 5 mmol l − 1 immediately at reperfusion ) was used to rephosphorylate GSK - 3β / mTOR / P70S6K . digits below represent semiquantified optidensity of bands , control bands were defined as 1 . 00 .

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

(a) Akt/GSK-3β/mTOR/P70S6K phosphorylation assessed by western blots after thioperamide administration and OGD/R. LiCl (5 mmol l−1 immediately at reperfusion) was used to rephosphorylate GSK-3β/mTOR/P70S6K. The digits below represent the semiquantified optidensity of the bands, the control bands were defined as 1.00.


( b ) Representative western blots showing phosphorylation of Akt / GSK - 3β / mTOR / P70S6K signaling in H3R − / − mice after OGD / R injury . digits below represent semiquantified optidensity of bands , control bands were defined as 1 . 00 .

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(b) Representative western blots showing the phosphorylation of Akt/GSK-3β/mTOR/P70S6K signaling in H3R−/− mice after OGD/R injury. The digits below represent the semiquantified optidensity of the bands, the control bands were defined as 1.00.


( c ) Representative showing LC3 - positive autophagic vacuoles ( red ) with 4 ' , 6 - diamidino - 2 - phenylindole ( blue ) after administration of thioperamide LiCl with OGD / R ( scale bar , 10 μm ) .

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(c) Representative images showing LC3-positive autophagic vacuoles (red) with 4',6-diamidino-2-phenylindole staining (blue) after administration of thioperamide and LiCl with OGD/R (scale bar, 10 μm).


( d ) viability assessed by MTT when thioperamide LiCl were administered after OGD / R ( n = 7 , * * P0 . 01 , * * * P0 . 001 with of variances ( ANOVAs ) followed by Bonferroni / Dunn post hoc ) .

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(d) Cell viability assessed by MTT when thioperamide and LiCl were administered after OGD/R (n=7, **P0.01, ***P0.001 with analysis of variances (ANOVAs) followed by the Bonferroni/Dunn post hoc test).


( e ) Representative western blots bar graph showing expression of LC3 - I LC3 - II in steady - state autophagy autophagic flux under OGD / R injury when thioperamide ( THIO ) immepip ( IMME ) were administered ( n = 6 ; * * P0 . 01 , * * * P0 . 001 with ANOVAs followed by Bonferroni / Dunn post hoc ) . Data are presented as mean ± s . e . m . Full - blots are shown in Supplementary Fig . 11 .

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(e) Representative western blots and bar graph showing the expression of LC3-I and LC3-II in steady-state autophagy and autophagic flux under OGD/R injury when thioperamide (THIO) and immepip (IMME) were administered (n=6; **P0.01, ***P0.001 with ANOVAs followed by the Bonferroni/Dunn post hoc test). Data are presented as mean±s.e.m. Full-size blots are shown in Supplementary Fig. 11.


( a ) Representative immunohistochemical of LC3 ( green ) along with NeuN ( red ) in WT H3R − / − mice ( scale bar , 25 μm ) .

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(a) Representative immunohistochemical staining of LC3 (green) along with NeuN staining (red) in WT and H3R−/− mice (scale bar, 25 μm).


( b , c ) When thioperamide was administered in vivo , LC3 expression was assessed by western blot after I / R in WT mice ( b ) ( * P0 . 05 , * * * P0 . 001 ; n = 6 mice per condition , with of variances ( ANOVAs ) followed by Bonferroni / Dunn post hoc ) , in H3R − / − mice ( c ) ( * * * P0 . 001 versus H3R − / − sham group ; n = 5 - 6 mice per condition , with ANOVAs followed by Bonferroni / Dunn post hoc . ) Data are presented as mean ± s . e . m . Full - blots are shown in Supplementary Fig . 12 .

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(b,c) When thioperamide was administered in vivo, LC3 expression was assessed by western blot after I/R in WT mice (b) (*P0.05, ***P0.001; n=6 mice per condition, with analysis of variances (ANOVAs) followed by the Bonferroni/Dunn post hoc test), and in H3R−/− mice (c) (***P0.001 versus H3R−/− sham group; n=5-6 mice per condition, with ANOVAs followed by the Bonferroni/Dunn post hoc test.) Data are presented as mean±s.e.m. Full-size blots are shown in Supplementary Fig. 12.


( a ) Western blots showing inhibitory effect of 3 - MA on autophagy in I / R . Full - blots are shown in Supplementary Fig . 13 .

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(a) Western blots showing the inhibitory effect of 3-MA on autophagy in I/R. Full-size blots are shown in Supplementary Fig. 13.


( b ) TTC - stained sections showing infarct after administration of 3 - MA ( 100 nmol , i . c . v . , at reperfusion ) in WT H3R − / − mice ( scale bar , 5 mm ) , ( c ) bar graph showing infarct volume ( n = 6 - 8 mice per condition ; * P0 . 05 , * * * P0 . 001 , with of variances ( ANOVAs ) followed by Bonferroni / Dunn post hoc ) .

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(b) TTC-stained sections showing the infarct area after administration of 3-MA (100 nmol, i.c.v., at reperfusion) in WT and H3R−/− mice (scale bar, 5 mm), and (c) bar graph showing infarct volume (n=6-8 mice per condition; *P0.05, ***P0.001, with analysis of variances (ANOVAs) followed by the Bonferroni/Dunn post hoc test).


( d ) TTC - stained sections ( scale bar , 5 mm ) bar graph showing infarct after administration of thioperamide in Atg5 + / − mice ( n = 4 - 6 mice per condition ) .

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(d) TTC-stained sections (scale bar, 5 mm) and bar graph showing the infarct area after administration of thioperamide in Atg5+/−mice (n=4-6 mice per condition).


( e ) In cultured neurons , viability was tested by MTT after 3 - MA ( 2 . 5 mmol l − 1 at reperfusion ) thioperamide was administered in OGD / R ( n = 7 per condition ; * * * P0 . 001 , with ANOVAs followed by Bonferroni / Dunn post hoc ) .

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(e) In cultured neurons, viability was tested by MTT assay after 3-MA (2.5 mmol l−1 at reperfusion) and thioperamide was administered in OGD/R (n=7 per condition; ***P0.001, with ANOVAs followed by the Bonferroni/Dunn post hoc test).


( f ) In cultured neurons , viability was tested by MTT after siRNA for Atg7 OGD / R ( n = 7 per condition ; * * * P0 . 001 , with ANOVAs followed by Bonferroni / Dunn post hoc ) .

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(f) In cultured neurons, viability was tested by MTT after siRNA for Atg7 and OGD/R (n=7 per condition; ***P0.001, with ANOVAs followed by the Bonferroni/Dunn post hoc test).


( g ) Viability was tested by MTT on Atg5 + / + Atg5 − / − MEFs after thioperamide administration after OGD / R ( n = 7 per condition ; * * P0 . 01 , * * * P0 . 001 , with ANOVAs followed by Bonferroni / Dunn post hoc ) . Data are presented as mean ± s . e . m .

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(g) Viability was tested by MTT assay on Atg5+/+and Atg5−/− MEFs after thioperamide administration after OGD/R (n=7 per condition; **P0.01, ***P0.001, with ANOVAs followed by the Bonferroni/Dunn post hoc test). Data are presented as mean±s.e.m.


( a ) Representative co - immunoprecipitation results showing effect of thioperamide on interaction of H3R with CLIC4 during OGD / R . Full - blots are shown in Supplementary Fig . 14 .

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(a) Representative co-immunoprecipitation results showing the effect of thioperamide on the interaction of H3R with CLIC4 during OGD/R. Full-size blots are shown in Supplementary Fig. 14.


( b ) viability was tested by MTT after administration of Tat - H3RCT414 - 436 ( 10 − 6 mol l − 1 at reperfusion ) under OGD / R ( data are presented as mean ± s . e . m . ; n = 7 per condition ; * P0 . 05 , * * P0 . 01 , with of variances ( ANOVAs ) followed by Bonferroni / Dunn post hoc ) .

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(b) Cell viability was tested by MTT assay after administration of Tat-H3RCT414-436 (10−6 mol l−1 at reperfusion) under OGD/R (data are presented as mean±s.e.m.; n=7 per condition; *P0.05, **P0.01, with analysis of variances (ANOVAs) followed by the Bonferroni/Dunn post hoc test).


( c ) Representative western blots showing effect of Tat - H3RCT414 - 436 on phosphorylation of Akt / GSK - 3β / mTOR / P70S6K signalling LC3 expression in OGD / R . Full - blots are shown in Supplementary Fig . 14 .

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(c) Representative western blots showing the effect of Tat-H3RCT414-436 on phosphorylation of Akt/GSK-3β/mTOR/P70S6K signalling and LC3 expression in OGD/R. Full-size blots are shown in Supplementary Fig. 14.


( d ) Representative of effect of Tat - H3RCT414 - 436 on LC3 - positive autophagic ( red ) in 4 ' , 6 - diamidino - 2 - phenylindole - stained ( blue ) neurons after OGD / R ( scale bar , 10 μm ) .

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(d) Representative images of the effect of Tat-H3RCT414-436 on LC3-positive autophagic puncta (red) in 4',6-diamidino-2-phenylindole-stained (blue) neurons after OGD/R (scale bar,10 μm).


( e ) Western blots showing effect of siRNA for CLIC4 on phosphorylation of Akt / GSK - 3β / mTOR / P70S6K signalling LC3 expression in OGD / R . Full - blots are shown in Supplementary Fig . 14 .

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(e) Western blots showing the effect of siRNA for CLIC4 on phosphorylation of Akt/GSK-3β/mTOR/P70S6K signalling and LC3 expression in OGD/R. Full-size blots are shown in Supplementary Fig. 14.


( f ) Representative western blots bar graph showing expression of LC3 - I LC3 - II on steady - state autophagy autophagic flux under OGD / R when CLIC4 was knocked down ( data are presented as mean ± s . e . m . ; n = 6 ; * P0 . 05 , * * P0 . 01 ; , with ANOVAs followed by Bonferroni / Dunn post hoc ) . Full - blots are shown in Supplementary Fig . 14 .

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(f) Representative western blots and bar graph showing the expression of LC3-I and LC3-II on steady-state autophagy and autophagic flux under OGD/R when CLIC4 was knocked down (data are presented as mean±s.e.m.; n=6; *P0.05, **P0.01; , with ANOVAs followed by the Bonferroni/Dunn post hoc test). Full-size blots are shown in Supplementary Fig. 14.


( g ) viability was assessed by MTT after siRNA for CLIC4 under OGD / R ( data are presented as mean ± s . e . m . ; n = 8 per condition ; * * * P0 . 001 , with ANOVAs followed by Bonferroni / Dunn post hoc ) .

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(g) Cell viability was assessed by MTT assay after siRNA for CLIC4 under OGD/R (data are presented as mean±s.e.m.; n=8 per condition; ***P0.001, with ANOVAs followed by the Bonferroni/Dunn post hoc test).


Immunoblot showed HEK293 transfected for 48 hours with expression vectors for TCHP , FLAG - TCHP at N - C - terminal . total lysates were immunoprecipitated with anti - FLAG antibodies , blots were probed sequentially with anti - PCM1 , anti - FLAG , anti - ACTIN antibodies . IgG chains are indicated with Red Ponceau . input totals were analyzed by parallel immunoblotting as a control for level of expression .

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Immunoblot analysis showed HEK293 transfected for 48 hours with expression vectors for TCHP, and FLAG-TCHP at N- and C- terminal. total lysates were immunoprecipitated with anti-FLAG antibodies, and blots were probed sequentially with anti-PCM1, anti-FLAG, and anti-ACTIN antibodies. IgG light chains are indicated with Red Ponceau staining. The input totals were analyzed by parallel immunoblotting as a control for the level of expression.


Immunoblot showed HEK293 transfected for 48 hours with expression vectors for TCHP - FLAG C - terminal or constructs with deletions of coiled - coil domain 1 ( TCHP ∆ 1 ) 2 ( TCHP ∆ 2 ) , as indicated in scheme . total lysates were immunoprecipitated with anti - FLAG antibodies , blots were probed sequentially with anti - PCM1 , anti - FLAG , anti - ACTIN antibodies . IgG chains are indicated with Red Ponceau . input totals were analyzed by parallel immunoblotting as a control for level of expression .

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Immunoblot analysis showed HEK293 transfected for 48 hours with expression vectors for TCHP-FLAG C-terminal or constructs with deletions of the coiled-coil domain 1 (TCHP ∆1) and 2 (TCHP ∆2), as indicated in the scheme. total lysates were immunoprecipitated with anti-FLAG antibodies, and blots were probed sequentially with anti-PCM1, anti-FLAG, and anti-ACTIN antibodies. IgG light chains are indicated with Red Ponceau staining. The input totals were analyzed by parallel immunoblotting as a control for the level of expression.


Anti - PCM1 immunoprecipitation from HUVECs TCHP PCM1 immunoblot .

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Anti-PCM1 immunoprecipitation from HUVECs cells and TCHP and PCM1 immunoblot.


Co - localization of TCHP - V5 centriolar satellite . HUVECs were transduced with TCHP - V5 lentivirus stained for anti - PCM1 , anti - CEP290 , anti - CEP72 anti - V5 antibodies . Scale bars , 25 µm 5 µm in inset . Right panel : two - channel correlation of pixels corresponding to regions identified with TCHP - V5 satellite markers ( n = 90 ; Pearson colocalization coefficient ) .

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Co-localization of TCHP-V5 and centriolar satellite proteins. HUVECs were transduced with TCHP-V5 lentivirus and stained for anti-PCM1, anti-CEP290, anti-CEP72 and anti-V5 antibodies. Scale bars, 25 µm and 5 µm in the inset. Right panel: the two-channel intensity correlation of pixels corresponding to regions identified with TCHP-V5 and satellite markers (n=90 cells; Pearson colocalization coefficient).


TCHP knock - down or control were stained for anti - PCM1 antibody . Panel below : ( n = 50 ; unpaired t - ; * * p = 0 . 004 vs control ) . Scale bars , 50 µm 5 µm in inset .

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TCHP knock-down or control cells were stained for anti-PCM1 antibody. Panel below: quantification (n=50 cells; unpaired t-test; **p=0.004 vs control). Scale bars, 50 µm and 5 µm in the inset.


Control or TCHP knock - down HUVECs were subjected to cycloheximide ( CHX ) MG132 treatments for indicated number of hours before immunoblotting . PCM1 were analysed in insoluble fraction . Laminin has been used as a loading control . Below panel : of PCM1 degradation . ( one - way ANOVA ; * p = 0 . 0180 vs control time 0 ) .

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Control or TCHP knock-down HUVECs were subjected to cycloheximide (CHX) and MG132 treatments for the indicated number of hours before immunoblotting. PCM1 levels were analysed in the insoluble fraction. Laminin has been used as a loading control. Below panel: Quantification of PCM1 degradation. (one-way ANOVA; *p=0.0180 vs control time 0).


Control or TCHP knock - down HUVECs were subjected to cycloheximide ( CHX ) MG132 treatments for indicated number of hours before immunoblotting . Condition as in ( A ) , Western blot for anti - GABARAP anti - ACTIN antibodies . Below panel : of GABARAP degradation . ( one - way ANOVA ; * p = 0 . 0215 vs control time 0 ) .

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Control or TCHP knock-down HUVECs were subjected to cycloheximide (CHX) and MG132 treatments for the indicated number of hours before immunoblotting. Condition as in (A), Western blot for anti-GABARAP and anti-ACTIN antibodies. Below panel: Quantification of GABARAP degradation. (one-way ANOVA; *p =0.0215 vs control time 0).


Control or TCHP overexpressing HUVECs were subjected to CHX treatment for indicated number of hours prior to immunoblotting . Western blot was probed for anti - PCM1 anti - V5 antibodies . Laminin has been used as a loading control .

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Control or TCHP overexpressing HUVECs were subjected to CHX treatment for the indicated number of hours prior to immunoblotting. Western blot was probed for anti-PCM1 and anti-V5 antibodies. Laminin has been used as a loading control.


Control or TCHP overexpressing HUVECs were subjected to CHX treatment for indicated number of hours prior to immunoblotting . Western blot was probed for anti - V5 , anti - GABARAP anti - ACTIN antibodies . Below panels : of C ( one - way ANOVA ; * * p < 0 . 0001 vs control time 0 ; # # p = 0 . 0003 vs control CHX ) D ( one - way ANOVA ; * * p < 0 . 0001 vs control time 0 ; # # p = 0 . 0056 vs control CHX ) .

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Control or TCHP overexpressing HUVECs were subjected to CHX treatment for the indicated number of hours prior to immunoblotting. Western blot was probed for anti-V5, anti-GABARAP and anti-ACTIN antibodies. Below panels: Quantification of C (one-way ANOVA; **p<0.0001 vs control time 0; ##p=0.0003 vs control CHX) and D (one-way ANOVA; **p<0.0001 vs control time 0; ##p=0.0056 vs control CHX).


Immunofluorescent for LC3 p62 in TCHP knock - down control . Scale bars , 25μm . Lower panel : . ( n = 90 , unpaired t - ; LC3 : * * p < 0 . 0001 vs control ; p62 : * * p < 0 . 0001 vs control ) .

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Immunofluorescent staining for LC3 and p62 in TCHP knock-down and control cells. Scale bars, 25μm. Lower panel: quantification. (n=90 cells, unpaired t-test; LC3: **p<0.0001 vs control; p62: **p<0.0001 vs control).


Western blot of p62 LC3 during under normal culture condition or starved condition ( HBSS ) or presence of BafA1 ( 2 hours ) in TCHP knock - down or control . Lower panels : p62 . ( one - way ANOVA ; * * p = 0 . 0003 vs control , # # p = 0 . 0093 vs shTCHP ) ; LC3 . ( one - way ANOVA ; * * p = 0 . 0008 vs control , # # p = 0 . 0011 vs shTCHP ) .

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Western blot of p62 and LC3 during under normal culture condition or starved condition (HBSS) or the presence of BafA1 (2 hours) in TCHP knock-down or control cells. Lower panels: p62 quantification. (one-way ANOVA; **p=0.0003 vs control, ##p=0.0093 vs shTCHP); LC3 quantification. (one-way ANOVA; **p=0.0008 vs control, ##p=0.0011 vs shTCHP).


HUVECs were transduced with tandem mCherry - EGFP - LC3 with shRNA TCHP or control vectors . Left panels : Representative picture of mCherrry - EGFP - LC3 reporters . Scale bars , 25μm . Right panel : of number of mCherry - only ( red bars , autolysosomes ) or double - positive ( mCherry + / EGFP + ; yellow bars , autophagosomes ) . ( n = 80 , one - way ANOVA ; * p = 0 . 0447 vs control ; # # p = 0 . 0007 vs shTCHP ) .

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HUVECs were transduced with the tandem mCherry-EGFP-LC3 and with shRNA TCHP or control vectors. Left panels: Representative picture of mCherrry-EGFP-LC3 reporters. Scale bars, 25μm. Right panel: Quantification of the number of mCherry-only (red bars, autolysosomes) or double-positive (mCherry+/EGFP+; yellow bars, autophagosomes). (n=80 cells, one-way ANOVA; *p=0.0447 vs control; ##p=0.0007 vs shTCHP).


Ratiometric flow cytometric of mCherrry - EGFP - LC3 reporters as in C . ( one - way ANOVA ; * p = 0 . 0247 vs control ; # # p = 0 . 0088 vs shTCHP ) .

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Ratiometric flow cytometric analysis of mCherrry-EGFP-LC3 reporters as in C. (one-way ANOVA; *p=0.0247 vs control; ##p=0.0088 vs shTCHP).


of long - lived degradation in TCHP knock - down control by flow cytometry ( one - way ANOVA ; * * p = 0 . 0018 vs control , # # p < 0 . 0001 vs shTCHP ) .

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Quantification of long-lived protein degradation assay in TCHP knock-down and control cells by flow cytometry (one-way ANOVA; **p=0.0018 vs control, ##p<0.0001 vs shTCHP).


HUVECs were transduced with TCHP - V5 control vectors . Western blot for anti - V5 anti - p62 antibody during under normal culture condition or HBSS or BafA1 or Torin - 1 treatment . Lower panel : . ( one - way ANOVA ; * * p = 0 . 0044 ; # p = 0 . 0117 vs TCHP - V5 ) .

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HUVECs were transduced with TCHP-V5 and control vectors. Western blot for anti-V5 and anti-p62 antibody during under normal culture condition or HBSS or BafA1 or Torin-1 treatment. Lower panel: quantification. (one-way ANOVA; **p=0.0044; #p=0.0117 vs TCHP-V5).


postnuclear fraction ( PNS ) from TCHP knock - down control HUVECs in presence or absence of BafA1 was separated into ( low - speed pellet ) LSP ( high - speed pellet ) HSP fractions then analysed by immunoblots using anti p62 , LC3 GABARAP antibodies . sub - fractions were treated with proteinase K ( Prot . K ) with or without Triton X - 100 ( TX - 100 ) . of Western blot : p62 , LC3 GABARAP relative to respective GAPDH were quantified using densitometry normalized to value of non - treated samples .

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The postnuclear fraction (PNS) from TCHP knock-down and control HUVECs in the presence or absence of BafA1 was separated into (low-speed pellet) LSP and (high-speed pellet) HSP fractions and then analysed by immunoblots using anti p62, LC3 and GABARAP antibodies. The sub-fractions were treated with proteinase K (Prot. K) with or without Triton X- 100 (TX-100). Quantification of the Western blot: the p62, LC3 and GABARAP levels relative to respective GAPDH were quantified using densitometry analysis and normalized to the value of non-treated samples.


Representative of autophagosomes in control TCHP knock - down HUVECs in complete medium after 3h incubation with BafA1 ( Scale bars , 600nm ) : i , of mean autophagic vesicles ( AVs ) autophagosome ( APs ) ii , distribution of cross - section areas of analysed vesicles expressed in . ( n = 7 ; > 100 vesicles per sample ; AVs : unpaired t - ; * * p < 0 . 0001 vs control ; APs : unpaired t - * * p < 0 . 0001 vs control ) .

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Representative TEM images of autophagosomes in control and TCHP knock-down HUVECs in the complete medium after 3h incubation with BafA1 (Scale bars, 600nm): i, quantification of mean autophagic vesicles (AVs) and autophagosome (APs) area and ii, distribution of the cross-section areas of the analysed vesicles expressed in percentage. (n=7 cells; >100 vesicles per sample; AVs: unpaired t-test; **p<0.0001 vs control; APs: unpaired t-test **p<0.0001 vs control).


Left panels : Representative single - channel merged of HUVECs expressing GFP - LC3 immunostained for STX17 . Scale bars , 25 µm 2 µm in inset . Right panels : of LC3 / STX17 positive in presence or absence of BafA1 ( one - way ANOVA ; * * p = 0 . 0060 vs control ; # p = 0 . 0136 vs control BafA1 ) LC3 / STX17 positive per ( n = 50 ; one - way ANOVA ; * * p < 0 . 0001 vs shTCHP BafA1 ) .

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Left panels: Representative single-channel and merged images of HUVECs expressing GFP-LC3 and immunostained for STX17. Scale bars, 25 µm and 2 µm in the inset. Right panels: Quantification of LC3/STX17 positive cells in the presence or absence of BafA1 (one-way ANOVA; **p=0.0060 vs control; #p=0.0136 vs control BafA1) and quantification LC3/STX17 positive puncta per cells (n=50 cells; one-way ANOVA; **p<0.0001 vs shTCHP BafA1).