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

O O O O O O B-SUBCELLULAR O O O O O O O O B-SUBCELLULAR O O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O O O B-CELL_LINE O O O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O B-SUBCELLULAR O B-SUBCELLULAR O O O O O O O O

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


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

O O O O O O B-SUBCELLULAR O O O O O O B-GENEPROD O O O B-CELL_LINE O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O

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


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

O O O O O O O B-SUBCELLULAR B-EXP_ASSAY O O O B-GENEPROD O O O B-CELL_LINE O O O O O O O O O B-GENEPROD O B-GENEPROD O O B-GENEPROD O O B-GENEPROD O O O O O O O O O O

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


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

O O B-EXP_ASSAY I-EXP_ASSAY O B-SUBCELLULAR B-EXP_ASSAY O O B-CELL_LINE O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O B-EXP_ASSAY O B-SMALL_MOLECULE O O O O O B-EXP_ASSAY O O O B-GENEPROD O O O O O

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


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

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


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

O O O O O O O B-SUBCELLULAR B-EXP_ASSAY O O B-CELL_LINE O O O O O O B-GENEPROD O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O

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


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

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


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

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


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

O O O O O O O B-SUBCELLULAR B-EXP_ASSAY O B-GENEPROD O O O B-CELL_LINE O O O O O O B-GENEPROD O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O

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


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

O O B-EXP_ASSAY I-EXP_ASSAY O B-SUBCELLULAR B-EXP_ASSAY O B-GENEPROD O O O B-CELL_LINE O O O O O O O O O O B-GENEPROD O O O O B-EXP_ASSAY O B-SMALL_MOLECULE O O O O

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


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

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


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

O O O O O O O O O O O O O O O O O B-SUBCELLULAR B-EXP_ASSAY O B-GENEPROD O O O B-CELL_LINE O O O O O O O O O O B-GENEPROD O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O

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


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

O O B-GENEPROD B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O O O O B-GENEPROD O O O O O O O O O O O O O O B-GENEPROD O O O B-CELL_LINE O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O B-EXP_ASSAY O O O O

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


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

B-GENEPROD B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O B-CELL_LINE O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O

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


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

O O B-GENEPROD B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O B-CELL_LINE O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O

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


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

O O O O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O O O O O B-CELL_LINE O O O O O O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O B-EXP_ASSAY O O O O

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


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

O O B-EXP_ASSAY O B-GENEPROD O O O B-GENEPROD O O O B-GENEPROD O O B-CELL_LINE O O O O O O B-GENEPROD O O O O O O O O O O B-SMALL_MOLECULE O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O B-EXP_ASSAY O O O O

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


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

O O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O O B-CELL_LINE O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O B-SMALL_MOLECULE O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O B-GENEPROD O O O O B-EXP_ASSAY O O O O

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


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

O O B-EXP_ASSAY O O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O O O B-CELL_LINE O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O B-GENEPROD O O O B-GENEPROD O O O O B-EXP_ASSAY O O O B-EXP_ASSAY I-EXP_ASSAY O O O O

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


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

O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-SUBCELLULAR O O O B-CELL_LINE O O O O O O B-SMALL_MOLECULE O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O B-GENEPROD O O B-CELL_LINE O O O B-SUBCELLULAR O B-SUBCELLULAR O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O B-SMALL_MOLECULE O O O O B-SUBCELLULAR O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-SUBCELLULAR O B-SUBCELLULAR O O O O O O B-SUBCELLULAR O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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


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

O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-SUBCELLULAR O O O B-CELL_LINE O O O O B-SUBCELLULAR O B-GENEPROD O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O B-GENEPROD O O B-CELL_LINE O O O O O B-SUBCELLULAR O B-GENEPROD O O O O O O O B-SUBCELLULAR O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O 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-SUBCELLULAR O O B-SUBCELLULAR O B-GENEPROD O O O O O O O O O O O O O O B-GENEPROD O O O O B-SUBCELLULAR O O B-SUBCELLULAR O B-GENEPROD O O O O O O O O O O O O O O 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-SUBCELLULAR O O O O O O O O O O O B-SUBCELLULAR O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O

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


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

O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-SUBCELLULAR O O O B-CELL_LINE O O O O O O B-SMALL_MOLECULE O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O B-GENEPROD O O O B-CELL_LINE O O O B-SUBCELLULAR O B-SUBCELLULAR O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O

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


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

O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-SUBCELLULAR O O O B-CELL_LINE O O O O B-SUBCELLULAR O B-GENEPROD O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O B-GENEPROD O O O O B-GENEPROD O O O O O O O O B-SUBCELLULAR O B-GENEPROD O B-SUBCELLULAR O B-GENEPROD O O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O

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


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

O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-SUBCELLULAR O O B-GENEPROD O O O B-CELL_LINE O O O O O O B-SMALL_MOLECULE O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O B-SUBCELLULAR O B-SUBCELLULAR O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O B-SMALL_MOLECULE O O O O O O O B-EXP_ASSAY O O O B-GENEPROD O O O O O B-SUBCELLULAR O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-SUBCELLULAR O B-SUBCELLULAR O O O O O O B-SUBCELLULAR O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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


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

O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-SUBCELLULAR O O B-GENEPROD O O O B-CELL_LINE O O O O B-SUBCELLULAR O B-GENEPROD O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O B-SUBCELLULAR O B-GENEPROD O B-SUBCELLULAR O B-GENEPROD O O O O O O O O O O O B-GENEPROD O O O O O O 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-SUBCELLULAR O O B-SUBCELLULAR O B-GENEPROD O O O O O O O O O O O O O B-GENEPROD O O O O B-SUBCELLULAR O O B-SUBCELLULAR O B-GENEPROD O O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O B-SUBCELLULAR O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O

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


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

O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-SUBCELLULAR O O B-GENEPROD O O O B-CELL_LINE O O O O O O B-SMALL_MOLECULE O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O B-SUBCELLULAR O B-SUBCELLULAR O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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


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

O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-SUBCELLULAR O O B-GENEPROD O O O B-CELL_LINE O O O O B-SUBCELLULAR O B-GENEPROD O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O B-GENEPROD O O O O B-GENEPROD O O O O O O O O B-SUBCELLULAR O B-GENEPROD O B-SUBCELLULAR O B-GENEPROD O O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O B-SUBCELLULAR O B-GENEPROD O O O O O O O O O O

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


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

O O O O B-SMALL_MOLECULE B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O B-GENEPROD O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O B-CELL_LINE O O O O O O B-GENEPROD O O O B-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O B-GENEPROD O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O

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


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

O O O O B-SMALL_MOLECULE B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O O O B-CELL_LINE O O O O O O O O O O O B-GENEPROD O O B-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O B-GENEPROD O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O B-EXP_ASSAY O O O B-GENEPROD O O O O O

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


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

O O O O B-SMALL_MOLECULE B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O B-CELL_LINE O O O O O O O O O O O O O B-GENEPROD O O B-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O O O B-GENEPROD O O O B-EXP_ASSAY O O O B-GENEPROD O O O O O

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


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

O O O O O O O O O O O O O O B-GENEPROD O O B-GENEPROD O O B-SUBCELLULAR B-EXP_ASSAY O B-GENEPROD O O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR B-EXP_ASSAY O B-GENEPROD O O O B-CELL_LINE O O O O B-GENEPROD O B-GENEPROD O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O

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


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

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


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

O O B-EXP_ASSAY I-EXP_ASSAY O B-SUBCELLULAR B-EXP_ASSAY O B-GENEPROD O O O O O O O O B-GENEPROD O B-GENEPROD B-CELL_LINE O O O O O O O O O O O B-EXP_ASSAY O B-EXP_ASSAY O B-SUBCELLULAR O O O O O O O O O O B-SUBCELLULAR O O

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


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

O O B-EXP_ASSAY I-EXP_ASSAY O B-SUBCELLULAR B-EXP_ASSAY O B-GENEPROD O B-GENEPROD B-CELL_LINE O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O B-SUBCELLULAR B-EXP_ASSAY O B-GENEPROD O O O B-CELL_LINE O O B-GENEPROD O B-GENEPROD O O O O O O O

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


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

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


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

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


Schematic diagram of ATXN8OS ( top strand ) and ATXN8 ( bottom strand ) . RNA - seq read coverage visualized by Integrative Genomic Viewer across the ATXN8 / ATXN8OS locus is depicted .

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Schematic diagram of ATXN8OS (top strand) and ATXN8 (bottom strand). RNA-seq read coverage visualized by Integrative Genomic Viewer across the ATXN8/ATXN8OS locus is depicted.


B Bar graph showing relative mRNA levels of ATXN8 and ATXN8OS . FPKM values for each transcript are normalized to FPKM value for ATXN8OS . ( * * * * ; p < 0 . 0001 ; mean ± SEM ; unpaired t test ) FPKM = fragments per kilo base of transcript per million mapped reads .

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B Bar graph showing relative mRNA levels of ATXN8 and ATXN8OS. FPKM values for each transcript are normalized to FPKM value for ATXN8OS. (****; p<0.0001; mean ± SEM; unpaired t test) FPKM= fragments per kilo base of transcript per million mapped reads.


D Immunohistochemistry of 10 - 12 month old end - stage SCA8 BAC mice brain tissue shows accumulation of novel polySer RAN protein ( detected by unique antibody to the polySer protein C - terminus , α - SerCT ) in cerebellar white matter , brainstem , hippocampus and layer II of the frontal cortex . Representative polySer aggregates are indicated by black arrows . No aggregates are found in age matched non - transgenic ( NT ) littermates ( n = 6 for each cohort ) .

O B-EXP_ASSAY O O O O O O O O O B-DISEASE O B-ORGANISM B-TISSUE O O O O O B-SMALL_MOLECULE O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O B-TISSUE I-TISSUE I-TISSUE O B-TISSUE O B-TISSUE O B-TISSUE I-TISSUE I-TISSUE I-TISSUE I-TISSUE I-TISSUE O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O B-ORGANISM O O O O O O O O O

D Immunohistochemistry of 10-12 month old end-stage SCA8 BAC mice brain tissue shows accumulation of novel polySer RAN protein (detected by unique antibody to the polySer protein C-terminus, α-SerCT) in cerebellar white matter, brainstem, hippocampus and layer II of the frontal cortex. Representative polySer aggregates are indicated by black arrows. No aggregates are found in age matched non-transgenic (NT) littermates (n=6 for each cohort).


F Immunohistochemistry of human SCA8 autopsy tissue ( n = 4 - 7 ) shows accumulation of novel polySer RAN protein in cerebellar white matter , brainstem and frontal cortex .

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F Immunohistochemistry of human SCA8 autopsy tissue (n=4-7) shows accumulation of novel polySer RAN protein in cerebellar white matter, brainstem and frontal cortex.


A Immunohistochemistry ( IHC ) of end - stage SCA8 BAC mouse cerebellum shows that polySer but not polyGln is found in the molecular layer and cerebellar white matter and that polyGln but not polySer accumulates in Purkinje cells . Representative polySer aggregates are indicated by black arrows . Inset : higher magnification of molecular layer and white matter .

O B-EXP_ASSAY O B-EXP_ASSAY O O O O O B-DISEASE O B-ORGANISM B-TISSUE O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O O O O B-TISSUE I-TISSUE O B-TISSUE I-TISSUE I-TISSUE O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O O B-TISSUE I-TISSUE O O B-SMALL_MOLECULE O O O O O O O O O O O O B-TISSUE I-TISSUE O B-TISSUE I-TISSUE O

A Immunohistochemistry (IHC) of end-stage SCA8 BAC mouse cerebellum shows that polySer but not polyGln is found in the molecular layer and cerebellar white matter and that polyGln but not polySer accumulates in Purkinje cells. Representative polySer aggregates are indicated by black arrows. Inset: higher magnification of molecular layer and white matter.


B IF double staining shows no co - localization of polyGln and polySer in frontal cortex , pons or hippocampus of end - stage SCA8 BAC mice .

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B IF double staining shows no co-localization of polyGln and polySer in frontal cortex, pons or hippocampus of end-stage SCA8 BAC mice.


C IF double staining of end - stage SCA8 BAC frontal cortex shows exclusive localization of polyGln ( mouse α - Gln , red , bottom panel ) in neurons ( rabbit α - NeuN , green , bottom panel ) . In contrast , polySer ( Rabbit α - SerCT , red , top panel ) shows widespread accumulation in the frontal cortex including within neurons ( mouse α - NeuN , green , top panel ) .

O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O B-DISEASE O B-TISSUE I-TISSUE O O B-EXP_ASSAY O B-SMALL_MOLECULE O B-ORGANISM O O O O O O O O O O B-CELL_TYPE O B-ORGANISM O O O O O O O O O O O O O B-SMALL_MOLECULE O B-ORGANISM O O O O O O O O O O O O O O B-TISSUE I-TISSUE O O B-CELL_TYPE O B-ORGANISM O O O O O O O O O O

C IF double staining of end-stage SCA8 BAC frontal cortex shows exclusive localization of polyGln (mouse α-Gln, red, bottom panel) in neurons (rabbit α-NeuN, green, bottom panel). In contrast, polySer (Rabbit α-SerCT, red, top panel) shows widespread accumulation in the frontal cortex including within neurons (mouse α-NeuN, green, top panel).


D IHC of SCA8 human cerebellum shows that PolySer accumulates in the white matter but not in the Purkinje cells ( left panels ) while polyGln accumulates in Purkinje cells but not cerebellar white matter ( right panels ) .

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D IHC of SCA8 human cerebellum shows that PolySer accumulates in the white matter but not in the Purkinje cells (left panels) while polyGln accumulates in Purkinje cells but not cerebellar white matter (right panels).


A Representative images of the vestibular nucleus ( upper panels ) , cuneate nucleus ( middle panels ) and motor cortex layers II / III ( lower panels ) of SCA8 BAC mice at 2 months ( left panels ) , 6 months ( middle panels ) and end - stage ( 10 months , right panels ) stained with α - SerCT . Representative aggregates are indicated by black arrows . Corresponding quantifications of polySer aggregates for each region on the right ( n = 3 ; mean ± SEM ; One - way ANOVA with Tukey ' s post - hoc test ; * p < 0 . 025 , * * p < 0 . 005 , * * * p < 0 . 0005 , * * * * p < 0 . 0001 )

O O O O O B-TISSUE I-TISSUE O O O O O B-TISSUE I-TISSUE O O O O O B-TISSUE I-TISSUE I-TISSUE I-TISSUE O O O O O O O B-DISEASE O B-ORGANISM O 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-EXP_ASSAY O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A Representative images of the vestibular nucleus (upper panels), cuneate nucleus (middle panels) and motor cortex layers II/III (lower panels) of SCA8 BAC mice at 2 months (left panels), 6 months (middle panels) and end-stage (10 months, right panels) stained with α-SerCT. Representative aggregates are indicated by black arrows. Corresponding quantifications of polySer aggregates for each region on the right (n=3; mean ± SEM; One-way ANOVA with Tukey's post-hoc test; * p<0.025, ** p<0.005, *** p<0.0005, **** p<0.0001)


B Open field analysis of 2 month old animals show a significant decrease in ambulatory distance ( cm ) and ambulatory time ( s ) and a significant increase in resting time ( s ) ( NT n = 19 , SCA8 BAC n = 23 , * * p < 0 . 01 , * * * p < 0 . 001 ; mean ± SEM ; unpaired t test ) .

O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O 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-DISEASE O O O O O O O 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 Open field analysis of 2 month old animals show a significant decrease in ambulatory distance (cm) and ambulatory time (s) and a significant increase in resting time (s) (NT n=19, SCA8 BAC n=23, ** p<0.01, *** p<0.001; mean ± SEM; unpaired t test).


A Disease - specific sensitivity to vacuolization after prolonged storage in ethanol shown by H & E in cerebellar white matter and brainstem of 10 - 12 month old end - stage SCA8 BAC mice but not in age matched NT ( n = 3 ) ( panels 1 & 2 ) . Demyelination shown by luxol fast blue staining ( LFB ) ( panel 3 ) and axonal degeneration shown by α - SMI - 32 ( panel 4 ; degenerated axons are indicated by black arrows ) observed in sites of polySer accumulation shown by α - SerCT ( panel 5 ; representative aggregates are indicated by black arrows ) in deep cerebellar white matter and brainstem in SCA8 BAC mice ( lower panels ) but not in NT mice ( upper panels ) ( n = 3 ) .

O O O O O O B-EXP_ASSAY O O O O B-SMALL_MOLECULE O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-TISSUE I-TISSUE I-TISSUE O B-TISSUE O O O O O O O O O B-DISEASE O B-ORGANISM O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-SMALL_MOLECULE O O O O O O B-SUBCELLULAR B-EXP_ASSAY O O O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O B-TISSUE I-TISSUE I-TISSUE O B-TISSUE O B-DISEASE O B-ORGANISM O O O O O O O O B-ORGANISM O O O O O O O O O O

A Disease-specific sensitivity to vacuolization after prolonged storage in ethanol shown by H&E in cerebellar white matter and brainstem of 10-12 month old end-stage SCA8 BAC mice but not in age matched NT (n=3) (panels 1&2). Demyelination shown by luxol fast blue staining (LFB) (panel 3) and axonal degeneration shown by α-SMI-32 (panel 4; degenerated axons are indicated by black arrows) observed in sites of polySer accumulation shown by α-SerCT (panel 5; representative aggregates are indicated by black arrows) in deep cerebellar white matter and brainstem in SCA8 BAC mice (lower panels) but not in NT mice (upper panels) (n=3).


Demyelination shown by luxol fast blue staining ( LFB ) ( panel 1 ) and axonal degeneration shown by α - SMI - 32 ( panel 2 ) observed sites of polySer accumulation shown by a - SerCT2 ( panel 3 ) in deep cerebellar white matter was found in SCA8 autopsy tissue but not in control brains ( n = 3 ) .

B-EXP_ASSAY O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-SMALL_MOLECULE O O O O O O B-SUBCELLULAR B-EXP_ASSAY O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O B-TISSUE I-TISSUE I-TISSUE I-TISSUE O O O B-DISEASE O O O O O O B-TISSUE O O O O O O

Demyelination shown by luxol fast blue staining (LFB) (panel 1) and axonal degeneration shown by α-SMI-32 (panel 2) observed sites of polySer accumulation shown by a-SerCT2 (panel 3) in deep cerebellar white matter was found in SCA8 autopsy tissue but not in control brains (n=3).


C Immunohistochemistry using CC1 ( α - APC ) antibody shows significantly lower numbers of mature oligodendrocytes in SCA8 BAC mice compared to NT mice ( NT n = 5 , SCA8 BAC n = 5 ; * * * * p < 0 . 0001 ; mean ± SEM ; unpaired t test ) .

O B-EXP_ASSAY O O O O O B-GENEPROD O O O O O O O B-CELL_TYPE I-CELL_TYPE O B-DISEASE O B-ORGANISM O O O B-ORGANISM O O O O O O B-DISEASE 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 Immunohistochemistry using CC1 (α- APC) antibody shows significantly lower numbers of mature oligodendrocytes in SCA8 BAC mice compared to NT mice (NT n=5, SCA8 BAC n=5; **** p<0.0001; mean ± SEM; unpaired t test).


D Immunofluorescence using α - GFAP antibody shows significant increase in astrogliosis in SCA8 BAC mice compared to NT mice ( NT n = 3 , SCA8 BAC n = 3 , * * p < 0 . 01 ; mean ± SEM ; unpaired t test ) .

O B-EXP_ASSAY O O O B-GENEPROD O O O O O B-EXP_ASSAY O B-DISEASE O B-ORGANISM O O O B-ORGANISM O O O O O O B-DISEASE O O O O O O O O O O O O O O O O O O O O O O

D Immunofluorescence using α-GFAP antibody shows significant increase in astrogliosis in SCA8 BAC mice compared to NT mice (NT n=3, SCA8 BAC n=3, ** p<0.01; mean ± SEM; unpaired t test).


E Cell death measured by lactase dehydrogenase ( LDH ) assay in T98 cells transfected with codon replacement constructs expressing polyGln ( light blue ) or polySer ( dark blue ) . NT = nontransfected , EV = empty vector ( n = 5 , * * p < 0 . 003 ; * * * p < 0 . 0003 ; mean ± SEM ; unpaired t test ) .

O B-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD I-GENEPROD B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-CELL_LINE O O O O O O O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O 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 Cell death measured by lactase dehydrogenase (LDH) assay in T98 cells transfected with codon replacement constructs expressing polyGln (light blue) or polySer (dark blue). NT=nontransfected, EV=empty vector (n=5, ** p<0.003; *** p< 0.0003; mean ± SEM; unpaired t test).


qRT - PCR showing Eif3f expression levels are increased 2 - fold in SCA8 cerebellar white matter compared to SCA8 cerebellar grey matter ( n = 3 , * * p < 0 . 01 ; mean ± SEM ; unpaired t test ) .

B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD B-EXP_ASSAY I-EXP_ASSAY O O O O O O B-DISEASE B-TISSUE I-TISSUE I-TISSUE O O B-DISEASE B-TISSUE I-TISSUE I-TISSUE O O O O O O O O O O O O O O O O O O O O O O

qRT-PCR showing Eif3f expression levels are increased 2-fold in SCA8 cerebellar white matter compared to SCA8 cerebellar grey matter (n=3, **p<0.01; mean ± SEM; unpaired t test).


C Dot blot detection of polySer expressionin using α - FLAG antibody showing decrease in RAN polySer but not ATG - polySer when HEK293 cells are co - transfected with eIF3F siRNA . D Quantification of polySer detection ( n = 5 , * p < 0 . 01 ; n . s . no significance ; mean ± SEM ; unpaired t test ) .

O B-EXP_ASSAY I-EXP_ASSAY O O B-SMALL_MOLECULE B-EXP_ASSAY O O O B-GENEPROD O O O O O B-SMALL_MOLECULE O O O O B-SMALL_MOLECULE O B-CELL_LINE O O O O O O B-GENEPROD O O O O O B-SMALL_MOLECULE O O O O O 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 Dot blot detection of polySer expressionin using α-FLAG antibody showing decrease in RAN polySer but not ATG-polySer when HEK293 cells are co-transfected with eIF3F siRNA. D Quantification of polySer detection (n=5, * p<0.01; n.s. no significance; mean ± SEM; unpaired t test).


E Detection of polyAla expression using α - HA antibody showing decrease in RAN polyAla but not ATG - polyAla when HEK293 cells are co - transfected with eIF3F siRNA . F Detection of polyGP expression using α - FLAG antibody showing a decrease in RAN polyGP when HEK293 cells are co - transfected with eIF3F siRNA . . G Quantification of polyAla and polyGP detection ( n = 5 , * p < 0 . 01 ; n . s . no significance ; mean ± SEM ; unpaired t test ) .

O O O B-SMALL_MOLECULE B-EXP_ASSAY O O O B-GENEPROD O O O O O B-SMALL_MOLECULE O O O O B-SMALL_MOLECULE O B-CELL_LINE O O O O O O B-GENEPROD O O O O O B-SMALL_MOLECULE B-EXP_ASSAY O O O B-GENEPROD O O O O O O B-SMALL_MOLECULE O B-CELL_LINE O O O O O O B-GENEPROD O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O 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 Detection of polyAla expression using α-HA antibody showing decrease in RAN polyAla but not ATG-polyAla when HEK293 cells are co-transfected with eIF3F siRNA. F Detection of polyGP expression using α-FLAG antibody showing a decrease in RAN polyGP when HEK293 cells are co-transfected with eIF3F siRNA.. G Quantification of polyAla and polyGP detection (n=5, * p<0.01; n.s. no significance; mean ± SEM; unpaired t test).


( A and B ) FACS - sorted T cell populations and Monocyte - derived Macrophages were treated with the indicated stimuli : Nigericin ( Nig . 4h ) , NeedleTox ( 4h ) , Val - boroPro ( VbP 22h ) , ABT737 / S63845 ( A / S 22h ) . When indicated , cells were primed with LPS for 2 hours prior to stimulation . When indicated , MCC950 was added to the media 30 minutes prior to the addition of Nigericin . LDH - activity ( A ) and IL - 1β and IL - 18 concentration ( B and C , respectively ) in the supernatant were determined by LDH cytotoxicity assay and ELISA respectively . Individual data points ± SEM from three independent donors are shown . Statistics indicate significance by two - way ANOVA : * * * p ≤ 0 . 001 ; * * p ≤ 0 . 01 ; * p ≤ 0 . 05 ; ns , not significant . P - values were corrected for multiple comparisons ( Dunnett ) .

O O O O O B-EXP_ASSAY O O B-CELL_TYPE I-CELL_TYPE O O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O B-SMALL_MOLECULE O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O B-SMALL_MOLECULE O B-GENEPROD O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O B-GENEPROD B-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A and B) FACS-sorted T cell populations and Monocyte-derived Macrophages were treated with the indicated stimuli: Nigericin (Nig. 4h), NeedleTox (4h), Val-boroPro (VbP 22h), ABT737/S63845 (A/S 22h). When indicated, cells were primed with LPS for 2 hours prior to stimulation. When indicated, MCC950 was added to the media 30 minutes prior to the addition of Nigericin. LDH-activity (A) and IL-1β and IL-18 concentration (B and C, respectively) in the supernatant were determined by LDH cytotoxicity assay and ELISA respectively. Individual data points ± SEM from three independent donors are shown. Statistics indicate significance by two-way ANOVA: ***p ≤ 0.001; **p ≤ 0.01; *p ≤ 0.05; ns, not significant. P-values were corrected for multiple comparisons (Dunnett).


A ) Macrophages and CD4 T cells from the same donor were subjected to the indicated treatments and morphologic changes as well as PI - uptake were monitored by live - cell imaging microscopy using a 25x objective . Representative images from indicated time points are shown . Cyan color coding is used for the fluorescent PI - signal . One donor out of two is shown . ( B ) Representative images were acquired with a 63x objective at the end of the experiment shown in ( A ) at 16 hours . One donor out of two is shown . Scale bars : 25 µm .

O O B-CELL_TYPE O B-GENEPROD B-CELL_TYPE I-CELL_TYPE O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Macrophages and CD4 T cells from the same donor were subjected to the indicated treatments and morphologic changes as well as PI-uptake were monitored by live-cell imaging microscopy using a 25x objective. Representative images from indicated time points are shown. Cyan color coding is used for the fluorescent PI-signal. One donor out of two is shown. (B) Representative images were acquired with a 63x objective at the end of the experiment shown in (A) at 16 hours. One donor out of two is shown. Scale bars: 25 µm.


( C ) CD4 T cells were treated with VbP or ABT737 / S63845 . Lysate and supernatant samples were collected at the indicated time points . Samples were analyzed by immunoblotting . αSS and αLS indicate the use of a small subunit or large subunit - specific caspase - 1 antibody respectively . ( Lys = lysate , SN = supernatant , FL = full length ) . One representative experiment out of three is shown .

O O O B-GENEPROD B-CELL_TYPE I-CELL_TYPE O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O 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) CD4 T cells were treated with VbP or ABT737/S63845. Lysate and supernatant samples were collected at the indicated time points. Samples were analyzed by immunoblotting. αSS and αLS indicate the use of a small subunit or large subunit-specific caspase-1 antibody respectively. (Lys = lysate, SN = supernatant, FL = full length). One representative experiment out of three is shown.


( A ) Gene expression profile of 15 inflammasome - related genes in human T cell and monocyte subsets . TPM values were retrieved from a public RNASeq dataset and log2 transformed . Both , rows and columns are clustered using euclidean distance and complete linkage . ( Tfh , T follicular helper cells ; Tregs , T regulatory cells ; Th , T helper cells ; CM , central memory T cells ; EM , effector memory T cells ; TE , terminal effector T cells ; CD14 + C16 - , classical monocytes ; CD14 + CD16 + , intermediate monocytes ; CD14 - CD16 + , non - classical monocytes ) .

(A) Gene expression profile of 15 inflammasome-related genes in human T cell and monocyte subsets. TPM values were retrieved from a public RNASeq dataset and log2 transformed. Both, rows and columns are clustered using euclidean distance and complete linkage. (Tfh, T follicular helper cells; Tregs, T regulatory cells; Th, T helper cells; CM, central memory T cells; EM, effector memory T cells; TE, terminal effector T cells; CD14+ C16-, classical monocytes; CD14+ CD16+, intermediate monocytes; CD14- CD16+, non-classical monocytes).


( B ) Immunoblotting of CD4 T cells and MDMs treated with Pam3CSK4 for 6 hours . * indicates an unspecific band . One representative experiment out of three is shown .

O O O B-EXP_ASSAY O B-GENEPROD B-CELL_TYPE I-CELL_TYPE O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O

(B) Immunoblotting of CD4 T cells and MDMs treated with Pam3CSK4 for 6 hours. * indicates an unspecific band. One representative experiment out of three is shown.


( C ) Immunoblotting of polyclonal CD4 T cells targeted with two gRNAs for each indicated target gene . One representative experiment out of three is shown .

O O O B-EXP_ASSAY O O B-GENEPROD B-CELL_TYPE I-CELL_TYPE O O O O O O O O O O O O O O O O O O O

(C) Immunoblotting of polyclonal CD4 T cells targeted with two gRNAs for each indicated target gene. One representative experiment out of three is shown.


( D ) CD4 T cells from the indicated polyclonal gene targeting approach were treated with the indicated stimuli for 18 hours . Individual data points ± SEM from three independent donors are shown . Statistics indicate significance by two - way ANOVA : * * * p ≤ 0 . 001 ; ns , not significant . P - values were corrected for multiple comparisons ( Dunnett ) .

O O O B-GENEPROD B-CELL_TYPE I-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) CD4 T cells from the indicated polyclonal gene targeting approach were treated with the indicated stimuli for 18 hours. Individual data points ± SEM from three independent donors are shown. Statistics indicate significance by two-way ANOVA: ***p ≤ 0.001; ns, not significant. P-values were corrected for multiple comparisons (Dunnett).


( B ) MiSeq analysis of the indicated gene targeting approaches from samples collected at the indicated time points post editing . DPP9 and DPP8 were targeted with one gRNA / target gene , CARD8 was targeted with two gRNAs . On the day of nucleofection , T cells were activated with human T activator CD3 / CD28 beads and expanded in the presence of IL - 2 . On day 10 , cells were switched to IL - 7 / IL - 15 containing medium . ( C ) Fraction of wildtype and in - frame reads are depicted . One representative experiment out of three is shown .

O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O B-GENEPROD O O O O O O O O O O O O B-CELL_TYPE I-CELL_TYPE O O O B-ORGANISM O O B-GENEPROD O B-GENEPROD O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O 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) MiSeq analysis of the indicated gene targeting approaches from samples collected at the indicated time points post editing. DPP9 and DPP8 were targeted with one gRNA/target gene, CARD8 was targeted with two gRNAs. On the day of nucleofection, T cells were activated with human T activator CD3/CD28 beads and expanded in the presence of IL-2. On day 10, cells were switched to IL-7/IL-15 containing medium. (C) Fraction of wildtype and in-frame reads are depicted. One representative experiment out of three is shown.


( A ) CD4 T cells were activated with human T activator CD3 / CD28 beads ( bead : cell ratio 1 : 5 ) for 4 days . Controls were kept resting in the presence of IL - 7 and IL - 15 . On day 4 cells were stimulated as indicated for 18 hours and cytotoxicity was assessed by LDH - activity in the supernatant . Individual data points ± SEM from four independent donors . Statistics indicate significance by two - way ANOVA : * * * p ≤ 0 . 001 ; ns , not significant . P - values were corrected for multiple comparisons ( Sidak ) .

O O O B-GENEPROD B-CELL_TYPE I-CELL_TYPE O O O B-ORGANISM O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O B-EXP_ASSAY O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) CD4 T cells were activated with human T activator CD3/CD28 beads (bead:cell ratio 1:5) for 4 days. Controls were kept resting in the presence of IL-7 and IL-15. On day 4 cells were stimulated as indicated for 18 hours and cytotoxicity was assessed by LDH-activity in the supernatant. Individual data points ± SEM from four independent donors. Statistics indicate significance by two-way ANOVA: ***p ≤ 0.001; ns, not significant. P-values were corrected for multiple comparisons (Sidak).


( B ) Immunoblotting of CD4 T cell lysates treated as in ( A ) for 3 to 4 days . * indicates an unspecific band . ( C ) Quantification of immunoblots shown in ( B ) and additional donors ( n = 9 for all but GSDMD , GSDMD n = 8 , for one donor quantification of the GSDMD band was considered unreliable due to uneven substrate distribution ) . Individual data points ± SEM are shown . Statistics indicate significance by one - way ANOVA of log2 - transformed fold change values of band intensities ( activated / non - activated T cells ) normalized to β - Actin : * * * p ≤ 0 . 001 ; * p ≤ 0 . 05 ; ns , not significant . P - values were corrected for multiple comparisons ( Sidak ) . All band intensity ratios were tested against β - Actin . CARD8 full length was additionally tested against CARD8 cleaved .

O O O B-EXP_ASSAY O B-GENEPROD B-CELL_TYPE I-CELL_TYPE 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-EXP_ASSAY O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O 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-CELL_TYPE I-CELL_TYPE O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O 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-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O O O O O B-GENEPROD O O

(B) Immunoblotting of CD4 T cell lysates treated as in (A) for 3 to 4 days. * indicates an unspecific band. (C) Quantification of immunoblots shown in (B) and additional donors (n= 9 for all but GSDMD, GSDMD n=8, for one donor quantification of the GSDMD band was considered unreliable due to uneven substrate distribution). Individual data points ± SEM are shown. Statistics indicate significance by one-way ANOVA of log2-transformed fold change values of band intensities (activated/non-activated T cells) normalized to β-Actin: ***p ≤ 0.001; *p ≤ 0.05; ns, not significant. P-values were corrected for multiple comparisons (Sidak). All band intensity ratios were tested against β-Actin. CARD8 full length was additionally tested against CARD8 cleaved.


( E ) Representative western blot of scr or miR - 30c transfected TREx cells analysed for ORF65 expression with GAPDH as a loading control . Densitometry analysis performed on n = 3 . Data information : data are presented as mean ± SD . * * P < 0 . 01 , * * * P < 0 . 001 ( Unpaired Student ' s t - test ) . All repeats are biological .

O O O O B-EXP_ASSAY I-EXP_ASSAY O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-CELL_LINE O O O B-GENEPROD B-EXP_ASSAY O B-GENEPROD O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O 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) Representative western blot of scr or miR-30c transfected TREx cells analysed for ORF65 expression with GAPDH as a loading control. Densitometry analysis performed on n=3. Data information: data are presented as mean ± SD. **P<0.01, ***P<0.001 (Unpaired Student's t-test). All repeats are biological.


( F ) Representative western blot of scr or miR - 29b transfected TREx cells analysed for ORF65 expression with GAPDH as a loading control . Densitometry analysis performed on n = 3 . Data information : data are presented as mean ± SD . * * P < 0 . 01 , * * * P < 0 . 001 ( Unpaired Student ' s t - test ) . All repeats are biological .

(F) Representative western blot of scr or miR-29b transfected TREx cells analysed for ORF65 expression with GAPDH as a loading control. Densitometry analysis performed on n=3. Data information: data are presented as mean ± SD. **P<0.01, ***P<0.001 (Unpaired Student's t-test). All repeats are biological.


( C ) Representative western blot of ORF65 levels in scr and circHIPK3 KD cell lines with GAPDH as a loading control . Densitometry analysis of n = 3 western blots . Data information : data are presented as mean ± SD . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 ( Unpaired Student ' s t - test ) . All repeats are biological .

O O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O O O O O O O B-GENEPROD O O O O O B-EXP_ASSAY O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Representative western blot of ORF65 levels in scr and circHIPK3 KD cell lines with GAPDH as a loading control. Densitometry analysis of n=3 western blots. Data information: data are presented as mean ± SD. *P<0.05, **P<0.01, ***P<0.001 (Unpaired Student's t-test). All repeats are biological.


( B ) Representative western blot of DLL4 expression in TREx cells 0 and 24 hours post induction . GAPDH was used a loading control ( n = 3 ) . Data information : data are presented as mean ± SD . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 ( Unpaired Student ' s t - test ) . All repeats are biological .

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(B) Representative western blot of DLL4 expression in TREx cells 0 and 24 hours post induction. GAPDH was used a loading control (n=3). Data information: data are presented as mean ± SD. *P<0.05, **P<0.01, ***P<0.001 (Unpaired Student's t-test). All repeats are biological.


( D ) Representative western blot of DLL4 levels in scr and circHIPK3 stable TREx cells at 0 and 24 hours post induction with GAPDH as a loading control . Densitometry analysis of n = 3 . Data information : data are presented as mean ± SD . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 ( Unpaired Student ' s t - test ) . All repeats are biological .

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(D) Representative western blot of DLL4 levels in scr and circHIPK3 stable TREx cells at 0 and 24 hours post induction with GAPDH as a loading control. Densitometry analysis of n=3. Data information: data are presented as mean ± SD. *P<0.05, **P<0.01, ***P<0.001 (Unpaired Student's t-test). All repeats are biological.


( F ) Representative western blot of DLL4 levels in scr or miR - 30c transfected TREx cells at 0 , 24 and 48 hours post induction with GAPDH as a loading control . Densitometry analysis of n = 3 Data information : data are presented as mean ± SD . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 ( Unpaired Student ' s t - test ) . All repeats are biological .

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(F) Representative western blot of DLL4 levels in scr or miR-30c transfected TREx cells at 0, 24 and 48 hours post induction with GAPDH as a loading control. Densitometry analysis of n=3 Data information: data are presented as mean ± SD. *P<0.05, **P<0.01, ***P<0.001 (Unpaired Student's t-test). All repeats are biological.


( J ) Representative western blot of DLL4 levels in scr or miR - 30c antagomiR transfected TREx cells at 0 , 24 and 48 hours post induction with GAPDH as a loading control . Densitometry analysis of n = 3 . Data information : data are presented as mean ± SD . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 ( Unpaired Student ' s t - test ) . All repeats are biological .

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(J) Representative western blot of DLL4 levels in scr or miR-30c antagomiR transfected TREx cells at 0, 24 and 48 hours post induction with GAPDH as a loading control. Densitometry analysis of n=3. Data information: data are presented as mean ± SD. *P<0.05, **P<0.01, ***P<0.001 (Unpaired Student's t-test). All repeats are biological.


( B ) Representative western blot of DLL4 levels in scr and DLL4 KD TREx cells with GAPDH as a loading control , densitometry analysis is n = 3 . Data information : data are presented as mean ± SD . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 ( Unpaired Student ' s t - test ) . All repeats are biological .

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(B) Representative western blot of DLL4 levels in scr and DLL4 KD TREx cells with GAPDH as a loading control, densitometry analysis is n=3. Data information: data are presented as mean ± SD. *P<0.05, **P<0.01, ***P<0.001 (Unpaired Student's t-test). All repeats are biological.


( C ) Representative western blot of ORF65 levels at 0 , 24 and 48 hours post induction in scr and DLL4 KD TREx cells , GAPDH was used as a loading control and densitometry analysis performed on n = 3 . Data information : data are presented as mean ± SD . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 ( Unpaired Student ' s t - test ) . All repeats are biological .

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(C) Representative western blot of ORF65 levels at 0, 24 and 48 hours post induction in scr and DLL4 KD TREx cells, GAPDH was used as a loading control and densitometry analysis performed on n=3. Data information: data are presented as mean ± SD. *P<0.05, **P<0.01, ***P<0.001 (Unpaired Student's t-test). All repeats are biological.


A . Histograms of spindle angle of GFP - H2B U2OS cells transfected with either GAPDH or Spindly siRNA and filmed on rectangular micropatterns . Cells were filmed 48 hours post - transfection .

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A. Histograms of spindle angle of GFP-H2B U2OS cells transfected with either GAPDH or Spindly siRNA and filmed on rectangular micropatterns. Cells were filmed 48 hours post-transfection.


B . Mitotic timing of control - depleted U2OS cells or CLIP - 170 - depleted cells . CLIP - 170 - depeleted cells are split in two categories ; ' aligned ' and ' misaligned ' , relating to the observed chromosome misalignment

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B. Mitotic timing of control-depleted U2OS cells or CLIP-170-depleted cells. CLIP-170-depeleted cells are split in two categories; 'aligned' and 'misaligned', relating to the observed chromosome misalignment phenotype


C . The histograms of spindle angles for the two different categories of CLIP - 170 - depleted U2OS cells ( Left graph with alignment defect , right graph without alignment defect ) .

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C. The histograms of spindle angles for the two different categories of CLIP-170-depleted U2OS cells (Left graph with alignment defect, right graph without alignment defect).


D and E . Mitotic timing and spindle angle histograms of control ( GAPDH ) - depleted U2OS cells and CENP - E - depleted cells .

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D and E. Mitotic timing and spindle angle histograms of control (GAPDH)-depleted U2OS cells and CENP-E-depleted cells.


A . HeLa cell stably expressing GFP - LGN and transiently expressing RFP - H2B was treated with noscapine to induce chromosome misalignments . Cells were imaged every 5 minutes . The time is relative to NEB . The red arrows indicate cortical regions where the chromosomes are in close proximity to the cell boundaries , accompanied by the displacement of LGN from the cortex .

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A. HeLa cell stably expressing GFP-LGN and transiently expressing RFP-H2B was treated with noscapine to induce chromosome misalignments. Cells were imaged every 5 minutes. The time is relative to NEB. The red arrows indicate cortical regions where the chromosomes are in close proximity to the cell boundaries, accompanied by the displacement of LGN from the cortex.


B . Similar assay as described in supplementary figure 2D was carried out in the presence of a small molecule inhibitor against Aurora - B ( ZM447439 ) in combination with the proteasome inhibitor MG - 132 to keep cells arrested in mitosis and in B . with an inhibitor against Plk1 ( BI2536 ) . Drugs were added at the same timing as Hoechst , 30 minutes prior to imaging .

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B. Similar assay as described in supplementary figure 2D was carried out in the presence of a small molecule inhibitor against Aurora-B (ZM447439) in combination with the proteasome inhibitor MG-132 to keep cells arrested in mitosis and in B. with an inhibitor against Plk1 (BI2536). Drugs were added at the same timing as Hoechst, 30 minutes prior to imaging.


A . Immunofluorescence images and quantifications of KT - localized Plk1 in U2OS cells after PBIP1 depletion . Cells were either Mock transfected or with PBIP1 siRNA for 72 hours . Cells were treated with nocodazole for 1 hour and fixed . Cells were stained with the centromere marker ACA , Plk1 and DAPI .

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A. Immunofluorescence images and quantifications of KT-localized Plk1 in U2OS cells after PBIP1 depletion. Cells were either Mock transfected or with PBIP1 siRNA for 72 hours. Cells were treated with nocodazole for 1 hour and fixed. Cells were stained with the centromere marker ACA, Plk1 and DAPI.


B . Representative images and histograms showing the effect of chromosome position on cortical LGN enrichment after 72 hours of Mock and PBIP1 depletion .

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B. Representative images and histograms showing the effect of chromosome position on cortical LGN enrichment after 72 hours of Mock and PBIP1 depletion.


C . Live cellimages and histograms of GFP - H2B U2OS cells Mock transfected or with siPBIP1 filmed on rectangular micropatterns in the presence of a CENP - E inhibitor . The spindle angles for both conditions were scored at 32 minutes after NEB .

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C. Live cellimages and histograms of GFP-H2B U2OS cells Mock transfected or with siPBIP1 filmed on rectangular micropatterns in the presence of a CENP-E inhibitor. The spindle angles for both conditions were scored at 32 minutes after NEB.


. D . The total spindle angle displacement within the first hour of mitosis was determined by measuring the spindle angle at each time point and calculating the sum of the Δ spindle angle between each time points .

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. D. The total spindle angle displacement within the first hour of mitosis was determined by measuring the spindle angle at each time point and calculating the sum of the Δ spindle angle between each time points.


C . RADX interacts with ssDNA . Extracts of U2OS cells stably expressing GFP - RADX WT or mutants were incubated with biotin - coupled ssDNA oligo immobilized on Streptavidin beads , washed extensively and immunoblotted with GFP and RPA1 antibodies .

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C. RADX interacts with ssDNA. Extracts of U2OS cells stably expressing GFP-RADX WT or mutants were incubated with biotin-coupled ssDNA oligo immobilized on Streptavidin beads, washed extensively and immunoblotted with GFP and RPA1 antibodies.


D . ssDNA - bound Streptavidin beads incubated with cell extracts as in ( C ) were washed with buffer containing indicated salt concentrations prior to immunoblotting .

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D. ssDNA-bound Streptavidin beads incubated with cell extracts as in (C) were washed with buffer containing indicated salt concentrations prior to immunoblotting.


E . Lysates of untransfected HCT116 cells were incubated with immobilized ssDNA or dsDNA probes as in ( C ) and immunoblotted with RADX and RPA1 antibodies .

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E. Lysates of untransfected HCT116 cells were incubated with immobilized ssDNA or dsDNA probes as in (C) and immunoblotted with RADX and RPA1 antibodies.


F . Immunoblot analysis of RADX expression in human cell lines . See also Fig EV1B .

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F. Immunoblot analysis of RADX expression in human cell lines. See also Fig EV1B.


G . Representative images from in situ proximity ligation assays ( PLAs ) in BrdU - labeled U2OS and GFP / GFP - RADX cell lines ( Fig EV1E , F ) using GFP and BrdU antibodies under native conditions . Scale bar , 10 μm .

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G. Representative images from in situ proximity ligation assays (PLAs) in BrdU-labeled U2OS and GFP/GFP-RADX cell lines (Fig EV1E,F) using GFP and BrdU antibodies under native conditions. Scale bar, 10 μm.


I . Soluble and chromatin - enriched fractions of U2OS cell lines stably expressing GFP - RADX alleles were immunoblotted with indicated antibodies .

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I. Soluble and chromatin-enriched fractions of U2OS cell lines stably expressing GFP-RADX alleles were immunoblotted with indicated antibodies.


A . Immunoblot analysis of chromatin fractions of U2OS cells transfected with indicated siRNAs and treated or not with HU .

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A. Immunoblot analysis of chromatin fractions of U2OS cells transfected with indicated siRNAs and treated or not with HU.


B . U2OS cells treated with HU in the presence or absence of ATR inhibitor ( ATRi ) were fractionated and immunoblotted with indicated antibodies . Relative RADX levels on chromatin were quantified and normalized to histone H3 . See also Fig EV2A .

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B. U2OS cells treated with HU in the presence or absence of ATR inhibitor (ATRi) were fractionated and immunoblotted with indicated antibodies. Relative RADX levels on chromatin were quantified and normalized to histone H3. See also Fig EV2A.


C . Representative images of U2OS cells stably expressing indicated GFP - RADX alleles that were subjected to laser microirradiation , fixed with methanol / acetone immediately afterwards and immunostained with γH2AX antibody .

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C. Representative images of U2OS cells stably expressing indicated GFP-RADX alleles that were subjected to laser microirradiation, fixed with methanol/acetone immediately afterwards and immunostained with γH2AX antibody.


D . As in ( C ) , except cells were fixed with paraformaldehyde 20 min after DNA damage infliction and immunostained with PCNA antibody .

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D. As in (C), except cells were fixed with paraformaldehyde 20 min after DNA damage infliction and immunostained with PCNA antibody.


E . Representative images from PLAs with GFP and RPA2 antibodies in U2OS and U2OS / GFP - RADX cell lines labeled with EdU for 30 min and then fixed or exposed to HU or CPT for 4 h before fixation .

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E. Representative images from PLAs with GFP and RPA2 antibodies in U2OS and U2OS/GFP-RADX cell lines labeled with EdU for 30 min and then fixed or exposed to HU or CPT for 4 h before fixation.


A . Immunoblot analysis of HCT116 cells transfected with indicated RADX siRNAs and RADX knockout cell lines ( RADXΔ ) .

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A. Immunoblot analysis of HCT116 cells transfected with indicated RADX siRNAs and RADX knockout cell lines (RADXΔ).


C . Cells in ( A ) were labeled with consecutive pulses of CldU and IdU as shown in ( B ) . Replication fork speeds were calculated as length of labeled track divided by pulse time ( bars , mean ; n = 400 fibers , pooled from two independent experiments , per condition ) .

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C. Cells in (A) were labeled with consecutive pulses of CldU and IdU as shown in (B). Replication fork speeds were calculated as length of labeled track divided by pulse time (bars, mean; n=400 fibers, pooled from two independent experiments, per condition).


D . Bidirectional replication fork symmetry was calculated as percentage of shorter divided by longer tracks from ( C ) . Concordance is 100 % , representing equal rates of bidirectional elongation for both daughter forks ( bars , mean ; n = 50 bidirectional forks , pooled from two independent experiments , per condition ) .

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D. Bidirectional replication fork symmetry was calculated as percentage of shorter divided by longer tracks from (C). Concordance is 100%, representing equal rates of bidirectional elongation for both daughter forks (bars, mean; n=50 bidirectional forks, pooled from two independent experiments, per condition).


E . Proportion of stalled forks ( CldU - only tracks ) among DNA fibers from ( C ) ( bars , mean ; 200 fibers analyzed per condition ; n = 2 independent experiments ) .

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E. Proportion of stalled forks (CldU-only tracks) among DNA fibers from (C) (bars, mean; 200 fibers analyzed per condition; n=2 independent experiments).


F . Proportion of new origins ( IdU - only tracks ) among DNA fibers in ( C ) ( bars , mean ; 20 fields of view quantified per condition ; n = 2 independent experiments ) .

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F. Proportion of new origins (IdU-only tracks) among DNA fibers in (C) (bars, mean; 20 fields of view quantified per condition; n=2 independent experiments).


H . Proportion of stalled forks ( CldU - only tracks ) among DNA fibers from cells labeled as in ( G ) ( bars , mean ; 200 fibers analyzed per condition ; n = 2 independent experiments ) .

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H. Proportion of stalled forks (CldU-only tracks) among DNA fibers from cells labeled as in (G) (bars, mean; 200 fibers analyzed per condition; n=2 independent experiments).


I . Proportion of restarted forks ( CldU - and IdU - positive tracks ) among DNA fibers in ( G ) ( bars , mean ; 200 fibers analyzed per condition ; n = 2 independent experiments ) .

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I. Proportion of restarted forks (CldU- and IdU-positive tracks) among DNA fibers in (G) (bars, mean; 200 fibers analyzed per condition; n=2 independent experiments).


A . Replication fork speeds in U2OS or U2OS / GFP - RADX cells transfected with control ( − ) or RADX ( # 6 ) siRNA targeting the 3 ' UTR and labeled with CldU and IdU as in Fig 3B ( bars , mean ; n = 400 fibers , pooled from two independent experiments , per condition ) .

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A. Replication fork speeds in U2OS or U2OS/GFP-RADX cells transfected with control (−) or RADX(#6) siRNA targeting the 3'UTR and labeled with CldU and IdU as in Fig 3B (bars, mean; n=400 fibers, pooled from two independent experiments, per condition).


B . Fork symmetry in cells treated as in ( A ) ( bars , mean ; n = 50 bidirectional forks , pooled from two independent experiments , per condition ) .

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B. Fork symmetry in cells treated as in (A) (bars, mean; n=50 bidirectional forks, pooled from two independent experiments, per condition).


C . Proportion of stalled forks ( CldU - only tracks ) among DNA fibers in ( A ) ( bars , mean ; 200 fibers analyzed per condition ; n = 2 independent experiments ) .

C. Proportion of stalled forks (CldU-only tracks) among DNA fibers in (A) (bars, mean; 200 fibers analyzed per condition; n=2 independent experiments).


D . Proportion of new origins ( IdU - only tracks ) among DNA fibers in ( A ) ( bars , mean ; 20 fields of view quantified per condition ; n = 2 independent experiments ) .

D. Proportion of new origins (IdU-only tracks) among DNA fibers in (A) (bars, mean; 20 fields of view quantified per condition; n=2 independent experiments).


E . Parental U2OS and derivative lines stably expressing GFP - RADX alleles or all three RPA isoforms at near - endogenous levels ( U2OS / Super - RPA ) ( Fig EV4A ) were transfected with control ( − ) or RADX siRNA . Replication fork speeds were determined as in ( A ) ( bars , mean ; n = 400 fibers , pooled from two independent experiments , per condition ) .

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E. Parental U2OS and derivative lines stably expressing GFP-RADX alleles or all three RPA isoforms at near-endogenous levels (U2OS/Super-RPA) (Fig EV4A) were transfected with control (−) or RADX siRNA. Replication fork speeds were determined as in (A) (bars, mean; n=400 fibers, pooled from two independent experiments, per condition).


F . Replication fork speeds in HCT116 cells with indicated genotypes transfected with control ( − ) , RPA1 ( 0 . 2 nM concentration [ 17 ] ) or RAD51 siRNAs ( Fig EV4B ) and labeled with CldU and IdU as in Fig 3B ( bars , mean ; n = 400 fibers , pooled from two independent experiments , per condition ) .

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F. Replication fork speeds in HCT116 cells with indicated genotypes transfected with control (−), RPA1 (0.2 nM concentration [17]) or RAD51 siRNAs (Fig EV4B) and labeled with CldU and IdU as in Fig 3B (bars, mean; n=400 fibers, pooled from two independent experiments, per condition).


G . Fork symmetry among DNA fibers in ( F ) ( bars , mean ; n = 50 bidirectional forks , pooled from two independent experiments , per condition ) .

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G. Fork symmetry among DNA fibers in (F) (bars, mean; n=50 bidirectional forks, pooled from two independent experiments, per condition).


H . New origin firing among DNA fibers in ( F ) ( bars , mean ; 20 fields of view quantified per condition ; n = 2 independent experiments ) .

H. New origin firing among DNA fibers in (F) (bars, mean; 20 fields of view quantified per condition; n=2 independent experiments).


B . Replication fork degradation ( IdU / CldU ratio ) in U2OS cells transfected with indicated siRNAs and processed as in ( A ) ( bars , mean ; n = 200 fibers , pooled from two independent experiments , per condition ) .

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B. Replication fork degradation (IdU/CldU ratio) in U2OS cells transfected with indicated siRNAs and processed as in (A) (bars, mean; n=200 fibers, pooled from two independent experiments, per condition).


C . As in ( B ) , except fork degradation was analyzed in U2OS / GFP - RADX cell lines .

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C. As in (B), except fork degradation was analyzed in U2OS/GFP-RADX cell lines.