B . Determination of the mtDNA copy number after H2O2 treatment . mtDNA / nDNA values in control , patient 1 and patient 2 fibroblasts . mtDNA : mitochondrial DNA , nDNA : nuclear DNA . C . mtDNA repair after H2O2 treatment . A long - range PCR was used to evaluate the oxidative damage , induced by H2O2 treatment , in mtDNA . The relative PCR amplification of a 15 . 6kb mtDNA fragment was normalized to relative PCR copy number that was evaluated by PCR amplification of a 172bp mtDNA fragment . mtDNA repair activity in control , patient 1 and patient 2 relative PCR . B - C . Cells were exposed to 150µM H2O2 for 30 min and either harvested immediately or allowed to recover in conditioned medium for the indicated times . Untreated control cultures were incubated in serum - free medium alone . Results represent the mean of relative PCR amplification ± SD of three independent experiments in which three PCRs per point were performed . Values were normalized to untreated cells and differences were analyzed by Student ' s t - test ( two - sided ) : significant ( * : 0 . 05 > p > 0 . 01 ) , very significant ( * * : 0 . 01 > p > 0 . 001 ) . Patient 1 versus control : * : p = 0 . 015 ( recovery 1h ) , * : p = 0 . 022 ( recovery 2h ) , * * : p = 0 . 003 ( recovery 4h ) . Patient 2 versus control : * : p = 0 . 0230 ( recovery 2h ) , * : p = 0 . 041 ( recovery 4h ) . NT : cells not treated with

O O O O O O O O O B-CONTROLLED_VAR O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR I-MEASURED_VAR O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Determination of the mtDNA copy number after H2O2 treatment. mtDNA/nDNA values in control, patient 1 and patient 2 fibroblasts. mtDNA : mitochondrial DNA, nDNA: nuclear DNA. C. mtDNA repair after H2O2 treatment. A long-range PCR was used to evaluate the oxidative damage, induced by H2O2 treatment, in mtDNA. The relative PCR amplification of a 15.6kb mtDNA fragment was normalized to relative PCR copy number that was evaluated by PCR amplification of a 172bp mtDNA fragment. mtDNA repair activity in control, patient 1 and patient 2 relative PCR. B-C. Cells were exposed to 150µM H2O2 for 30 min and either harvested immediately or allowed to recover in conditioned medium for the indicated times. Untreated control cultures were incubated in serum-free medium alone. Results represent the mean of relative PCR amplification ± SD of three independent experiments in which three PCRs per point were performed. Values were normalized to untreated cells and differences were analyzed by Student's t-test (two-sided): significant (*:0.05>p>0.01), very significant (**:0.01>p>0.001). Patient 1 versus control: *: p=0.015 (recovery 1h), *: p=0.022 (recovery 2h), **: p=0.003 (recovery 4h). Patient 2 versus control: *: p=0.0230 (recovery 2h), *: p=0.041 (recovery 4h). NT: cells not treated with H2O2


Figure 6 . No autophagy inhibition ( A - C ) and no mitophagy defect ( D - E ) in patient fibroblasts . A . Immunoblot of LC3B - I / - II expression in fibroblasts of patient 2 ( 1 , 2 ) , patient 1 ( 3 , 4 ) and control individual ( 5 , 6 ) in absence ( - ) or presence ( + ) of chloroquine ( CLQ ) . B . Quantitative analysis of LC3B - II / β - tubulin ratio from immunoblots ( n = 3 ) . C . Quantitative analysis of LC3B - II + CLQ / LC3B - II - CLQ ratio from immunoblots ( n = 3 ) .

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Figure 6. No autophagy inhibition (A-C) and no mitophagy defect (D-E) in patient fibroblasts. A. Immunoblot of LC3B-I/-II expression in fibroblasts of patient 2 (1,2), patient 1 (3,4) and control individual (5,6) in absence (-) or presence (+) of chloroquine (CLQ). B. Quantitative analysis of LC3B-II/β-tubulin ratio from immunoblots (n=3). C. Quantitative analysis of LC3B-II + CLQ/ LC3B-II - CLQ ratio from immunoblots (n=3).


D . Confocal images of fibroblasts derived from a control , patient 1 and patient 2 . Boxed regions are enlarged . Scale bar : 20µm . E . Bar graph showing ratios of fluorescent intensity of mitochondria delivered to lysosomes relative to that of mitochondria present in the cytosol ( FL lyso / FL mito ) . Statistical analysis were performed on mean ± SEM using Student ' s t - test ( two - sided ) .

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D. Confocal images of fibroblasts derived from a control, patient 1 and patient 2. Boxed regions are enlarged. Scale bar: 20µm. E. Bar graph showing ratios of fluorescent intensity of mitochondria delivered to lysosomes relative to that of mitochondria present in the cytosol (FL lyso/FL mito ). Statistical analysis were performed on mean ± SEM using Student's t-test (two-sided).


Figure 7 . CHCHD10 mutant fibroblasts are less sensitive to apoptotic cell death compared to control fibroblasts . A . Control ( C1 , C2 ) and patient ( P1 , P2 ) fibroblasts were treated with 1 μM of staurosporine ( STS ) for 16 h . Cell death was determined by flow cytometry using Annexin V / DAPI staining . Three independent experiments were performed per condition with two points analyzed per experiment . Differences between the control and patient fibroblasts were analyzed by Student ' s t - test ( two - sided ) : significant ( * : p = 0 . 0116 ) or extremely significant ( * * * : p = 0 . 0001 ) .

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Figure 7. CHCHD10 mutant fibroblasts are less sensitive to apoptotic cell death compared to control fibroblasts. A. Control (C1, C2) and patient (P1, P2) fibroblasts were treated with 1 μM of staurosporine (STS) for 16 h. Cell death was determined by flow cytometry using Annexin V/DAPI staining. Three independent experiments were performed per condition with two points analyzed per experiment. Differences between the control and patient fibroblasts were analyzed by Student's t-test (two-sided): significant (*: p=0.0116) or extremely significant (***: p=0.0001).


B - C . Fibroblasts were treated with 1 μM STS for 4 , 6 or 16 h ( O / N ) , and caspase activation was determined either by western blot ( B ) assessing the cleavage of PARP and the caspase 3 ( actin was used as a loading control )

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B-C. Fibroblasts were treated with 1 μM STS for 4, 6 or 16 h (O/N), and caspase activation was determined either by western blot (B) assessing the cleavage of PARP and the caspase 3 (actin was used as a loading control)


B - C . Fibroblasts were treated with 1 μM STS for 4 , 6 or 16 h ( O / N ) , and caspase activation was determined either by western blot ( B ) assessing the cleavage of PARP and the caspase 3 ( actin was used as a loading control ) or by DEVDase activity measurement ( C ) . Two ( B ) or three ( C ) independent experiments were performed per condition . Differences between the control and patient fibroblasts were analyzed by Student ' s t - test ( two - sided ) : extremely significant ( * * * : p = 0 . 0001 ) .

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B-C. Fibroblasts were treated with 1 μM STS for 4, 6 or 16 h (O/N), and caspase activation was determined either by western blot (B) assessing the cleavage of PARP and the caspase 3 (actin was used as a loading control) or by DEVDase activity measurement (C). Two (B) or three (C) independent experiments were performed per condition. Differences between the control and patient fibroblasts were analyzed by Student's t-test (two-sided): extremely significant (***: p=0.0001).


D - E . Mitochondrial membrane potential ( ΔΨM ) was measured by flow cytometry in control and patient fibroblasts after treatment of 1μM STS for 24 h ( E ) and quantified in D from two independent experiments . Differences between the control and patient fibroblasts were analyzed by Student ' s t - test ( two - sided ) : significant ( * : 0 . 05 > p > 0 . 01 ) . Patient 1 ( P1 ) versus control ( C2 ) : * : p = 0 . 0414 ( 24h ) . Patient 2 ( P2 ) versus control ( C2 ) : * : p = 0 . 0461 ( 24h ) .

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D-E. Mitochondrial membrane potential (ΔΨM) was measured by flow cytometry in control and patient fibroblasts after treatment of 1μM STS for 24 h (E) and quantified in D from two independent experiments. Differences between the control and patient fibroblasts were analyzed by Student's t-test (two-sided): significant (*:0.05>p>0.01). Patient 1 (P1) versus control (C2): *: p=0.0414 (24h). Patient 2 (P2) versus control (C2): *: p=0.0461 (24h).


Figure 8 . Overexpression of the CHCHD10 mutant alleles in HeLa cells leads to inhibition of apoptotic cell death . Transfections were performed with empty vector ( EV ) or vectors encoding either wild - type CHCHD10 - FLAG ( WT ) or mutant CHCHD10 - FLAG ( P34S and S59L ) . A . Western blot on HeLa cells extracts using antibodies against FLAG or Hsp90 .

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

Figure 8. Overexpression of the CHCHD10 mutant alleles in HeLa cells leads to inhibition of apoptotic cell death. Transfections were performed with empty vector (EV) or vectors encoding either wild-type CHCHD10-FLAG (WT) or mutant CHCHD10-FLAG (P34S and S59L). A. Western blot on HeLa cells extracts using antibodies against FLAG or Hsp90.


B - D . HeLa cells transiently expressing the WT or mutated forms of CHCHD10 - FLAG ( P34S or S59L ) were treated with 1 μM Actinomycine D ( ActD ) for 4 , 6 or 8 h with measurement of Annexin V / DAPI staining ( B ) , DEVD - ase activity ( C ) from three independent experiments . Differences between the mutated and non mutated alleles were analyzed by Student ' s t - test ( two - sided ) : significant ( * : 0 . 05 > p > 0 . 01 ) or very significant ( * * : 0 . 01 > p > 0 . 001 ) . P34S versus WT : * * : p = 0 . 0055 ( 4h ) , * : p = 0 . 0103 ( 8h ) . S59L versus WT : * : p = 0 . 0304 ( 4h ) , * : p = 0 . 0129 ( 8h ) .

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

B-D. HeLa cells transiently expressing the WT or mutated forms of CHCHD10-FLAG (P34S or S59L) were treated with 1 μM Actinomycine D (ActD) for 4, 6 or 8 h with measurement of Annexin V/DAPI staining (B), DEVD-ase activity (C) from three independent experiments. Differences between the mutated and non mutated alleles were analyzed by Student's t-test (two-sided): significant (*:0.05>p>0.01) or very significant (**:0.01>p>0.001). P34S versus WT: **: p=0.0055 (4h), *: p=0.0103 (8h). S59L versus WT: *: p=0.0304 (4h), *: p=0.0129 (8h).


D . Mitochondrial outer membrane permeabilization was determined by western blot by assessing the degradation of Smac . Hsp90 was used as a loading control .

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D. Mitochondrial outer membrane permeabilization was determined by western blot by assessing the degradation of Smac. Hsp90 was used as a loading control.


Bar graph represents IL - 2 secretion by NKT cell hybridoma ( 2C12 ) co - cultured with ( A ) a murine macrophage cell line ( J774 . 2 ) treated either with thapsigargin ( black bars ) , tunicamycin ( red bars ) or DMSO ( white bars ) . Graphs show mean ± SEM from n = 3biological replicates . * * * * P < 0 . 0001 ( Unpaired t test ) .

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Bar graph represents IL-2 secretion by NKT cell hybridoma (2C12) co-cultured with (A) a murine macrophage cell line (J774.2) treated either with thapsigargin (black bars),tunicamycin (red bars) or DMSO (white bars). Graphs show mean ± SEM from n=3biological replicates. ****P < 0.0001 (Unpaired t test).


Bar graph represents IL - 2 secretion by NKT cell hybridoma ( 2C12 ) co - cultured with a human macrophage cell line ( U937 ) , treated either with thapsigargin ( black bars ) , tunicamycin ( red bars ) or DMSO ( white bars ) . Graphs show mean ± SEM from n = 3biological replicates . * * * * P < 0 . 0001 ( Unpaired t test ) .

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

Bar graph represents IL-2 secretion by NKT cell hybridoma (2C12) co-cultured with a human macrophage cell line (U937), treated either with thapsigargin (black bars),tunicamycin (red bars) or DMSO (white bars). Graphs show mean ± SEM from n=3biological replicates. ****P < 0.0001 (Unpaired t test).


Heat map represents cytokine secretion by in vitro expanded ( C ) murine splenic iNKT cells co - cultured either with thapsigargin - or tunicamycin - or DMSO - treated murine BMDMs . The heat map shows the average result of two pooled biological replicates .

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Heat map represents cytokine secretion by in vitro expanded (C) murine splenic iNKT cells co-cultured either with thapsigargin- or tunicamycin- or DMSO-treated murine BMDMs. The heat map shows the average result of two pooled biological replicates.


Heat map represents cytokine secretion by in vitro expanded human peripheral blood iNKT cells co - cultured either with thapsigargin - or tunicamycin - or DMSO - treated murine BMDMs . The heat map shows the average result of two pooled biological replicates .

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Heat map represents cytokine secretion by in vitro expanded human peripheral blood iNKT cells co-cultured either with thapsigargin- or tunicamycin- or DMSO-treated murine BMDMs. The heat map shows the average result of two pooled biological replicates.


Wild type C57BL / 6 mice were injected intravenously with thapsigargin - loaded PLGA nanoparticles or vehicle , and 12 h later , hepatic iNKT cells were analysed by flow cytometry . Histograms represent expression of intracellular IL - 4 and IFN - γ levels in hepatic iNKT cells from mice injected with thapsigargin - loaded PLGA nanoparticles ( red histogram ) or vehicle - treated ( grey histogram ) mice . Dot plots represents MFI for intracellular IL - 4 and IFN - γ expression in hepatic iNKT cells from thapsigargin - loaded PLGA nanoparticles ( red dots ) or vehicle - treated ( black dots ) mice . Data plots show mean ± SEM from n = 5 mice per group . * * P ≤ 0 . 001 ( Mann - Whitney U test ) .

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Wild type C57BL/6 mice were injected intravenously with thapsigargin-loaded PLGA nanoparticles or vehicle, and 12 h later, hepatic iNKT cells were analysed by flow cytometry. Histograms represent expression of intracellular IL-4 and IFN-γ levels in hepatic iNKT cells from mice injected with thapsigargin-loaded PLGA nanoparticles (red histogram) or vehicle-treated (grey histogram) mice. Dot plots represents MFI for intracellular IL-4 and IFN-γ expression in hepatic iNKT cells from thapsigargin-loaded PLGA nanoparticles (red dots) or vehicle-treated (black dots) mice. Data plots show mean ± SEM from n = 5 mice per group. **P≤ 0.001 (Mann-Whitney U test).


Thapsigargin or αGalCer treated J774 . 2 cells were cocultured with 2C12 in the presence or absence of the murine CD1d - specific antibody ( black circles ) or isotype ( red squares ) for 16 hours . The addition of an anti - CD1d antibody blocked the activation of 2C12 demonstrating that the NKT - cell activation was CD1d - specific . Graphs show mean ± SEM from n = 2 biological replicates .

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

Thapsigargin or αGalCer treated J774.2 cells were cocultured with 2C12 in the presence or absence of the murine CD1d-specific antibody (black circles) or isotype (red squares) for 16 hours. The addition of an anti-CD1d antibody blocked the activation of 2C12 demonstrating that the NKT-cell activation was CD1d-specific. Graphs show mean ± SEM from n=2 biological replicates.


Bar graph represents IL - 2 secretion by NKT cell hybridoma ( 2C12 ) co - cultured murine macrophage cell line ( J774 . 2 ) treated either with thapsigargin - or tunicamycin - treated CD1d knockdown ( white bars ) or untransfected ( black bars ) . Knockdown of CD1d blocked IL - 2 secretion by 2C12 . In total , n = 2 biological replicates were performed ( three technical replicates per biological replicate ) . Graphs show mean ± SEM of each biological replicate .

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

Bar graph represents IL-2 secretion by NKT cell hybridoma (2C12) co-cultured murine macrophage cell line (J774.2) treated either with thapsigargin- or tunicamycin-treated CD1d knockdown (white bars) or untransfected (black bars). Knockdown of CD1d blocked IL-2 secretion by 2C12. In total, n=2 biological replicates were performed (three technical replicates per biological replicate). Graphs show mean ± SEM of each biological replicate.


Bar graph represents IL - 2 secretion by NKT cell hybridoma ( 2C12 ) co - cultured either with thapsigargin - or tunicamycin - treated primary BMDM isolated from CD1d - / - ( white bars ) or wild type ( black bars ) mice . 2C12 cocultured with CD1d - / - BMDMs resulted in reduced IL - 2 secretion . Graphs show mean ± SEM from n = 3 biological replicates . * * * P < 0 . 001 ( Unpaired t test ) .

Bar graph represents IL-2 secretion by NKT cell hybridoma (2C12) co-cultured either with thapsigargin- or tunicamycin-treated primary BMDM isolated from CD1d-/- (white bars) or wild type (black bars) mice. 2C12 cocultured with CD1d-/- BMDMs resulted in reduced IL-2 secretion. Graphs show mean ± SEM from n=3 biological replicates. ***P < 0.001 (Unpaired t test).


qPCR analyses showing the expression ( fold induction ) of UPR signature genes compared to DMSO in murine macrophage cell lines J774 . 2 stimulated either with thapsigargin , tunicamycin or DMSO for 4 or 8 hours . Graphs show mean ± SEM from n = 2 biological replicates ( two technical replicates per biological replicate ) .

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qPCR analyses showing the expression (fold induction) of UPR signature genes compared to DMSO in murine macrophage cell lines J774.2 stimulated either with thapsigargin, tunicamycin or DMSO for 4 or 8 hours. Graphs show mean ± SEM from n=2 biological replicates (two technical replicates per biological replicate).


qPCR analyses showing the expression ( fold induction ) of UPR signature genes compared to DMSO in human macrophage cell lines U937 stimulated either with thapsigargin , tunicamycin or DMSO for 4 or 8 hours . Graphs show mean ± SEM from n = 2 biological replicates ( two technical replicates per biological replicate ) .

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qPCR analyses showing the expression (fold induction) of UPR signature genes compared to DMSO in human macrophage cell lines U937 stimulated either with thapsigargin, tunicamycin or DMSO for 4 or 8 hours. Graphs show mean ± SEM from n=2 biological replicates (two technical replicates per biological replicate).


Bar graph represents percentage of IL - 2 secretion by NKT cell hybridoma ( 2C12 ) co - cultured with murine macrophage cell line ( J774 . 2 ) treated either with thapsigargin or tunicamycin in the presence of IRE1α inhibitor ( 4μ8c ) ( black bar ) or PERK inhibitor ( GSK ) ( grey bar ) or ATF6α inhibitor ( Ceapin - a7 ) ( red bar ) respectively . Inhibition of IRE1α or PERK resulted in significant reduction in IL - 2 secretion by iNKT hybridoma compared to controls . In total , n = 2 biological replicates were performed ( two and four technical replicates per biological replicate respectively ) . Graphs show mean ± SEM of each biological replicate .

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

Bar graph represents percentage of IL-2 secretion by NKT cell hybridoma (2C12) co-cultured with murine macrophage cell line (J774.2) treated either with thapsigargin or tunicamycin in the presence of IRE1α inhibitor (4μ8c) (black bar) or PERK inhibitor (GSK) (grey bar) or ATF6α inhibitor (Ceapin-a7) (red bar) respectively. Inhibition of IRE1α or PERK resulted in significant reduction in IL-2 secretion by iNKT hybridoma compared to controls. In total, n=2 biological replicates were performed (two and four technical replicates per biological replicate respectively). Graphs show mean ± SEM of each biological replicate.


Immunoblots represents the knockdown efficiency for IRE1α and PERK protein in J774 . 2 cell line transduced with lentiviral particles carrying gRNA against the IRE1α or PERK gene .

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Immunoblots represents the knockdown efficiency for IRE1α and PERK protein in J774.2 cell line transduced with lentiviral particles carrying gRNA against the IRE1α or PERK gene.


Bar graph represents percentage of IL - 2 secretion by NKT cell hybridoma ( 2C12 ) co - cultured with thapsigargin - treated murine IRE1α or PERK knockdown macrophage cell line ( J774 . 2 ) . Knockdown of IRE1α ( black bar ) or PERK ( grey bar ) resulted in significant reduction in IL - 2 secretion by iNKT hybridoma . In total n = 2 biological replicates were performed ( three technical replicates per biological replicate ) . Graphs show mean ± SEM of each biological replicate .

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

Bar graph represents percentage of IL-2 secretion by NKT cell hybridoma (2C12) co-cultured with thapsigargin-treated murine IRE1α or PERK knockdown macrophage cell line (J774.2). Knockdown of IRE1α (black bar) or PERK (grey bar) resulted in significant reduction in IL-2 secretion by iNKT hybridoma. In total n=2 biological replicates were performed (three technical replicates per biological replicate). Graphs show mean ± SEM of each biological replicate.


Neutral ( Fractions 1 - 3 ) and polar lipids ( Fractions 4 - 6 ) were isolated from J774 . 2 macrophages stimulated with thapsigargin , tunicamycin or DMSO and fractionated using solid - phase extraction ( SPE ) cartridges . Bar graph represents NKT cell activation by CD1d protein bound neutral lipids fractions isolated from thapsigargin - ( black bars ) or tunicamycin - treated ( red bars ) J774 . 2 cells . Polar lipid fractions induce very little IL - 2 secretion by 2C12 . In total , n = 2 biological replicates were performed ( two technical replicates per biological replicate ) . Graphs show mean ± SEM of each biological replicate .

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Neutral (Fractions 1-3) and polar lipids (Fractions 4-6) were isolated from J774.2 macrophages stimulated with thapsigargin, tunicamycin or DMSO and fractionated using solid-phase extraction (SPE) cartridges. Bar graph represents NKT cell activation by CD1d protein bound neutral lipids fractions isolated from thapsigargin- (black bars) or tunicamycin-treated (red bars) J774.2 cells. Polar lipid fractions induce very little IL-2 secretion by 2C12. In total, n=2 biological replicates were performed (two technical replicates per biological replicate). Graphs show mean ± SEM of each biological replicate.


Graph represents enhancement of loading with neutral lipid fractions isolated from thapsigargin - treated J774 . 2 at acidic pH and reduced mCD1d loading at neutral pH . Graphs show mean ± SEM from n = 2 biological replicates ( two technical replicates per biological replicate ) .

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Graph represents enhancement of loading with neutral lipid fractions isolated from thapsigargin-treated J774.2 at acidic pH and reduced mCD1d loading at neutral pH. Graphs show mean ± SEM from n=2 biological replicates (two technical replicates per biological replicate).


Neutral ( Fractions 1 - 3 ) and polar lipids ( Fractions 4 - 5 ) , isolated from murine bone marrow derived macrophages stimulated with thapsigargin or DMSO and fractionated using solid - phase extraction ( SPE ) cartridges , were co - cultured with purified NKT1 subsets . Bar graphs represent IFN - γ release by NKT1 cells induced by CD1d protein bound neutral lipids fractions isolated from thapsigargin - ( black bars ) or DMSO - treated ( white bars ) primary murine macrophages . Polar lipid fractions induce a minimal amount of IFN - γ secretion by the NKT1 sublineage . In total , n = 2 biological replicates were performed ( two technical replicates per biological replicate ) . Graphs shows mean ± SEM of each biological replicate .

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

Neutral (Fractions 1-3) and polar lipids (Fractions 4-5), isolated from murine bone marrow derived macrophages stimulated with thapsigargin or DMSO and fractionated using solid-phase extraction (SPE) cartridges, were co-cultured with purified NKT1 subsets. Bar graphs represent IFN-γ release by NKT1 cells induced by CD1d protein bound neutral lipids fractions isolated from thapsigargin- (black bars) or DMSO-treated (white bars) primary murine macrophages. Polar lipid fractions induce a minimal amount of IFN-γ secretion by the NKT1 sublineage. In total, n=2 biological replicates were performed (two technical replicates per biological replicate). Graphs shows mean ± SEM of each biological replicate.


Neutral ( Fractions 1 - 3 ) and polar lipids ( Fractions 4 - 5 ) were isolated from primary macrophages derived from human peripheral blood stimulated with thapsigargin or DMSO and fractionated using solid - phase extraction ( SPE ) cartridges . Bar graph represents IFN - γ release by NKT1 cells by CD1d protein bound neutral lipids fractions isolated from thapsigargin - ( black bars ) or DMSO - treated ( white bars ) human primary macrophages . Polar lipid fractions induce only minimal amounts of IFN - γ secretion by the NKT1 sublineage . In total , n = 2 biological replicates were performed ( two technical replicates per biological replicate ) . Graphs shows mean ± SEM of each biological replicate .

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

Neutral (Fractions 1-3) and polar lipids (Fractions 4-5) were isolated from primary macrophages derived from human peripheral blood stimulated with thapsigargin or DMSO and fractionated using solid-phase extraction (SPE) cartridges. Bar graph represents IFN-γ release by NKT1 cells by CD1d protein bound neutral lipids fractions isolated from thapsigargin- (black bars) or DMSO-treated (white bars) human primary macrophages. Polar lipid fractions induce only minimal amounts of IFN-γ secretion by the NKT1 sublineage. In total, n=2 biological replicates were performed (two technical replicates per biological replicate). Graphs shows mean ± SEM of each biological replicate.


Bar graph represents that recycling of CD1d between various endomembrane compartments and the cell surface is required for optimal IL - 2 secretion by NKT cells . In total , n = 2 biological replicates were performed ( three technical replicates per biological replicate ) . Graphs show mean ± SEM of each biological replicate .

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Bar graph represents that recycling of CD1d between various endomembrane compartments and the cell surface is required for optimal IL-2 secretion by NKT cells. In total, n=2 biological replicates were performed (three technical replicates per biological replicate). Graphs show mean ± SEM of each biological replicate.


qPCR analyses showing the expression ( fold induction ) of genes encoding for glycosphingolipid degrading enzymes compared to DMSO in murine macrophage cell lines J774 . 2 stimulated either with thapsigargin or DMSO for 8 hours . In total n = 2 biological replicates were performed ( two technical replicates per biological replicate ) . Graphs show mean ± SEM of each biological replicate .

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qPCR analyses showing the expression (fold induction) of genes encoding for glycosphingolipid degrading enzymes compared to DMSO in murine macrophage cell lines J774.2 stimulated either with thapsigargin or DMSO for 8 hours. In total n=2 biological replicates were performed (two technical replicates per biological replicate). Graphs show mean ± SEM of each biological replicate.


Glycolipid processing in intracellular compartments is not required prior to presentation to NKT cells . IL - 2 secretion in UPR - induced NKT cell activation in the presence or absence of castanospermine or deoxygalactonojirimycin . Graphs show mean ± SEM from n = 3 biological replicates . n . s ( non - significant ) ( One - way ANOVA ) biological replicates .

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Glycolipid processing in intracellular compartments is not required prior to presentation to NKT cells. IL-2 secretion in UPR-induced NKT cell activation in the presence or absence of castanospermine or deoxygalactonojirimycin. Graphs show mean ± SEM from n = 3 biological replicates. n.s (non-significant) (One-way ANOVA) biological replicates.


L363 antibody does not impact UPR - induced NKT cell activation . 2C12 NKT cells were co - cultured with ER - stressed macrophages J774 . 2 cells and treated with L363 antibody or isotype control . α - GalCer induced responses served as positive control . In total , n = 2 biological replicates were performed ( Four technical replicates per biological replicate ) . Graphs show mean ± SEM of each biological replicate .

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L363 antibody does not impact UPR-induced NKT cell activation. 2C12 NKT cells were co-cultured with ER-stressed macrophages J774.2 cells and treated with L363 antibody or isotype control. α-GalCer induced responses served as positive control. In total, n=2 biological replicates were performed (Four technical replicates per biological replicate). Graphs show mean ± SEM of each biological replicate.


Meth Induces Rapid Alkalization of Acidic Organelles , and Changes in Endosomal Structure in Dendritic Cells ( A ) Morphometric analysis of the decrease in acridine orange staining after 10 min treatment . Similar results were obtained in three independent experiments . ( Statistics n = 25 - 35 , mean ± SEM , * * p < 0 . 001 , * * * p < 0 . 0001 , two - tailed ANOVA . )

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Meth Induces Rapid Alkalization of Acidic Organelles, and Changes in Endosomal Structure in Dendritic Cells(A) Morphometric analysis of the decrease in acridine orange staining after 10 min treatment. Similar results were obtained in three independent experiments. (Statistics n = 25-35, mean ± SEM, **p < 0.001, ***p < 0.0001, two-tailed ANOVA.)


( B ) Progressive quenching of cytosolic acridine orange staining over 4 h in the absence ( ) or presence ( ▴ ) of 50 μM Meth . p < 0 . 001 for all time points versus control ( n = 25 - 35 , mean ± SEM , Student ' s two - tailed t - Test ) .

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

(B) Progressive quenching of cytosolic acridine orange staining over 4 h in the absence () or presence (▴) of 50 μM Meth. p < 0.001 for all time points versus control (n = 25-35, mean ± SEM, Student's two-tailed t-Test).


( C ) Acidic organelle pH in cells exposed to Meth for 10 min measured by ratiometric measurement . ( Statistics n = 30 - 90 cells , mean ± SEM , * * p < 0 . 001 , * * * p < 0 . 0001 , two - tailed ANOVA . )

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(C) Acidic organelle pH in cells exposed to Meth for 10 min measured by ratiometric measurement. (Statistics n = 30-90 cells, mean ± SEM, **p < 0.001, ***p < 0.0001, two-tailed ANOVA.)


( D ) Representative electron micrographs showing multivesicular endosomal compartments ( arrows ) in control , Clq - and Meth - treated cells at 4 h . Scale bar , 200 nm .

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(D) Representative electron micrographs showing multivesicular endosomal compartments (arrows) in control, Clq- and Meth-treated cells at 4 h. Scale bar, 200 nm.


( E ) Ultra - thin cryosections of control , Clq - and Meth - treated dendritic cells as in ( D ) , immunolabeled for LAMP - 1 ( 15 nm gold particles ) and MHC class II ( 10 nm gold particles ) . Scale bar , 200 nm .

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(E) Ultra-thin cryosections of control, Clq- and Meth-treated dendritic cells as in (D), immunolabeled for LAMP-1 (15 nm gold particles) and MHC class II (10 nm gold particles). Scale bar, 200 nm.


( A ) Accumulation of GFP - LC3 labeled autophagosomes in Meth - or Clq - treated dendritic cells at 2 h and 24 h . Fisher ' s exact test P values versus control ( n = 300 - 400 cells , mean ± SEM , * p < 0 . 05 , * * * p < 0 . 0001 ) .

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(A) Accumulation of GFP-LC3 labeled autophagosomes in Meth- or Clq-treated dendritic cells at 2 h and 24 h. Fisher's exact test P values versus control (n =300-400 cells, mean ± SEM, *p < 0.05, ***p < 0.0001).


( B ) GFP - LC3 labeled autophagosome formation in different concentrations of Meth after 24 h treatment . Similar results were obtained in three independent experiments . Fisher ' s exact test versus control ( n = 300 - 400 cells , mean ± SEM , * * * p < 0 . 0001 ) .

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(B) GFP-LC3 labeled autophagosome formation in different concentrations of Meth after 24 h treatment. Similar results were obtained in three independent experiments. Fisher's exact test versus control (n = 300-400 cells, mean ± SEM, ***p < 0.0001).


( C ) Representative fluorescence micrographs of GFP - LC3 decorated autophagosomes in cells treated with 50 μM Meth or 10 μM Clq for 24 h . Arrow denotes examples of autophagosomes . Scale bar , 10 μm .

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(C) Representative fluorescence micrographs of GFP-LC3 decorated autophagosomes in cells treated with 50 μM Meth or 10 μM Clq for 24 h. Arrow denotes examples of autophagosomes. Scale bar, 10 μm.


( A ) Cells were incubated in indicated concentrations of Meth or Clq for 2 h , and then IgG coated erythrocytes were added . Extracellular uningested EIgG were lysed and removed . Phagocytic index ( PI ) was quantified and inhibition is indicated as percent of control . Data were collected from four to seven experiments ( n = 300 cells , mean ± SEM , * * * p < 0 . 0001 , two - tailed ANOVA ) .

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(A) Cells were incubated in indicated concentrations of Meth or Clq for 2 h, and then IgG coated erythrocytes were added. Extracellular uningested EIgG were lysed and removed. Phagocytic index (PI) was quantified and inhibition is indicated as percent of control. Data were collected from four to seven experiments (n = 300 cells, mean ± SEM, ***p < 0.0001, two-tailed ANOVA).


( B ) Images of macrophage cells impaired in the phagocytosis of opsinized sheep erythrocytes after 2 h of Meth or Clq treatment . Arrows denote phagocytosed erythrocytes . Scale bar , 10 μm .

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(B) Images of macrophage cells impaired in the phagocytosis of opsinized sheep erythrocytes after 2 h of Meth or Clq treatment. Arrows denote phagocytosed erythrocytes. Scale bar, 10 μm.


( C , D ) J774 . 16 cells were exposed to PBS , chloroquine , or methamphetamine for 2 h followed by incubation with Cn or Ca . The phagocytic indices ( ratio of intracellular yeast to the number of macrophages counted ) were determined after 1 h or 30 min for Cn and Ca , respectively ( C ) . ( Statistics n = 300 , mean ± SEM , * p < 0 . 05 , two - tailed ANOVA . ) CFU determinations were preformed after 24 h incubations ( D ) . The values are the mean and standard deviation from determinations made in triplicate wells ( n = 300 cells , mean ± SEM , * p < 0 . 05 , two - tailed ANOVA . ) . Experiments were repeated three times with similar results .

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(C, D) J774.16 cells were exposed to PBS, chloroquine, or methamphetamine for 2 h followed by incubation with Cn or Ca. The phagocytic indices (ratio of intracellular yeast to the number of macrophages counted) were determined after 1 h or 30 min for Cn and Ca, respectively (C). (Statistics n = 300, mean ± SEM, *p < 0.05, two-tailed ANOVA.) CFU determinations were preformed after 24 h incubations (D). The values are the mean and standard deviation from determinations made in triplicate wells (n = 300 cells, mean ± SEM, *p < 0.05, two-tailed ANOVA.). Experiments were repeated three times with similar results.


A . Spindle in a fixed HeLa cell expressing PRC1 - GFP ( green ) and mRFP - CENP - B ( magenta ) oriented horizontally with respect to the imaging plane , as shown in the scheme . Images of different z - slices ( central plane of the spindle z = 0 , two images below , z = - 4 µm and z = - 2 µm , and above , z = + 3 µm and z = + 5 µm ) , maximum projection of a z - stack ( max z ) , and 3D projections ( 3D ) with the 3D coordinate system represented as a cuboidal box that indicates different spindle orientations are shown . Additional z - slices of this spindle are shown in Fig EV1A . B . Spindle in a fixed HeLa cell expressing PRC1 - GFP ( green ) and mRFP - CENP - B ( magenta ) oriented vertically with respect to the imaging plane , as shown in the scheme . Legend as in A . Additional z - slices of this spindle are shown in Fig EV1B . C . Correlation between the number of PRC1 - labeled bundles and the number of pairs of sister kinetochores counted throughout horizontal ( black ) and vertical ( blue ) spindles of fixed HeLa cells in metaphase . Data points represent individual spindles , lines show linear fits . D . Pie charts showing the fraction of PRC1 - labeled bundles associated with kinetochore pairs ( blue ) , PRC1 - labled bundles not associated with kinetochores ( green ) and kinetochores not associated with PRC1 bundles ( magenta ) in horizontal ( top row ) and vertical spindles ( bottom row ) from cells with up to 70 chromosomes ( left column ) and more than 70 chromosomes ( right column ) . Horizontal spindles contained a total of 1786 kinetochore - PRC1 pairs ( n = 29 cells ) and vertical spindles 1133 kinetochore - PRC1 pairs ( n = 16 cells ) . E , F . Spindle length and spindle width as a function of the number of kinetochore pairs coupled with PRC1 - labeled bundles . G . Number of kinetochore pairs coupled with PRC1 - labeled bundles per unit area of the cross section of the central part of horizontal ( black ) and vertical ( blue ) spindles as a function of the number of kinetochore pairs coupled with PRC1 - labeled bundles in the spindle .

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A. Spindle in a fixed HeLa cell expressing PRC1-GFP (green) and mRFP-CENP-B (magenta) oriented horizontally with respect to the imaging plane, as shown in the scheme. Images of different z-slices (central plane of the spindle z=0, two images below, z=-4 µm and z=-2 µm, and above, z=+3 µm and z=+5 µm), maximum projection of a z-stack (max z), and 3D projections (3D) with the 3D coordinate system represented as a cuboidal box that indicates different spindle orientations are shown. Additional z-slices of this spindle are shown in Fig EV1A.B. Spindle in a fixed HeLa cell expressing PRC1-GFP (green) and mRFP-CENP-B (magenta) oriented vertically with respect to the imaging plane, as shown in the scheme. Legend as in A. Additional z-slices of this spindle are shown in Fig EV1B.C. Correlation between the number of PRC1-labeled bundles and the number of pairs of sister kinetochores counted throughout horizontal (black) and vertical (blue) spindles of fixed HeLa cells in metaphase. Data points represent individual spindles, lines show linear fits.D. Pie charts showing the fraction of PRC1-labeled bundles associated with kinetochore pairs (blue), PRC1-labled bundles not associated with kinetochores (green) and kinetochores not associated with PRC1 bundles (magenta) in horizontal (top row) and vertical spindles (bottom row) from cells with up to 70 chromosomes (left column) and more than 70 chromosomes (right column). Horizontal spindles contained a total of 1786 kinetochore-PRC1 pairs (n=29 cells) and vertical spindles 1133 kinetochore-PRC1 pairs (n=16 cells).E, F. Spindle length and spindle width as a function of the number of kinetochore pairs coupled with PRC1-labeled bundles.G. Number of kinetochore pairs coupled with PRC1-labeled bundles per unit area of the cross section of the central part of horizontal (black) and vertical (blue) spindles as a function of the number of kinetochore pairs coupled with PRC1-labeled bundles in the spindle.


A . Central plane of the spindle in a live HeLa cell expressing PRC1 - GFP ( green ) and mRFP - CENP - B ( magenta ) oriented vertically with respect to the imaging plane . B . Examples of trajectories , with respect to the spindle ' s center of the mass , of individual PRC1 - GFP ( green ) and corresponding mRFP - CENP - B ( magenta ) signals from the spindle in A that moved together for at least 200 s . Dots represent starting points of trajectories , t = 0 s . Trajectories finish at t = 200 s . Grey circle represents the center of mass of the spindle . C . Pie chart showing the fraction of PRC1 - labeled fibers and sister kinetochores that moved together for at least 60 s ( blue , n = 226 pairs in 5 cells ) . A small fraction of PRC1 - labeled fibers ( green , n = 32 in 5 cells ) and kinetochores ( magenta , n = 16 in 5 cells ) did not move together .

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A. Central plane of the spindle in a live HeLa cell expressing PRC1-GFP (green) and mRFP-CENP-B (magenta) oriented vertically with respect to the imaging plane.B. Examples of trajectories, with respect to the spindle's center of the mass, of individual PRC1-GFP (green) and corresponding mRFP-CENP-B (magenta) signals from the spindle in A that moved together for at least 200 s. Dots represent starting points of trajectories, t=0 s. Trajectories finish at t=200 s. Grey circle represents the center of mass of the spindle.C. Pie chart showing the fraction of PRC1-labeled fibers and sister kinetochores that moved together for at least 60 s (blue, n=226 pairs in 5 cells). A small fraction of PRC1-labeled fibers (green, n=32 in 5 cells) and kinetochores (magenta, n=16 in 5 cells) did not move together.


D . Correlation coefficients between trajectories ( with respect to the spindle ' s center of the mass ) of a kinetochore pair and the trajectories of the PRC1 - labeled fiber coupled to the kinetochore pair ( green ) , the nearest neighbor PRC1 - labeled fiber ( blue ) , the next nearest neighbor ( black ) and a randomly chosen PRC1 - labeled fiber ( grey ) ; n , the number of trajectories from 3 cells .

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D. Correlation coefficients between trajectories (with respect to the spindle's center of the mass) of a kinetochore pair and the trajectories of the PRC1-labeled fiber coupled to the kinetochore pair (green), the nearest neighbor PRC1-labeled fiber (blue), the next nearest neighbor (black) and a randomly chosen PRC1-labeled fiber (grey); n, the number of trajectories from 3 cells.


E . Examples of scenarios observed in dynamic interplay of PRC1 - labeled bundles ( green ) and kinetochores ( magenta ) from the spindle in A . Row 1 : PRC1 - labeled bundle and kinetochore move together until they separate at 130 s . Row 2 : kinetochore is free of any PRC1 - labeled bundle until it appears at 130 s . Row 3 : kinetochore and PRC1 - labeled bundle move together . Row 4 : kinetochore moves with PRC1 - labeled bundle which merges with neighboring bundle at 169 s . Row 5 : two kinetochores move together with a single PRC1 - labeled bundle .

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E. Examples of scenarios observed in dynamic interplay of PRC1-labeled bundles (green) and kinetochores (magenta) from the spindle in A. Row 1: PRC1-labeled bundle and kinetochore move together until they separate at 130 s. Row 2: kinetochore is free of any PRC1-labeled bundle until it appears at 130 s. Row 3: kinetochore and PRC1-labeled bundle move together. Row 4: kinetochore moves with PRC1-labeled bundle which merges with neighboring bundle at 169 s. Row 5: two kinetochores move together with a single PRC1-labeled bundle.


A . Top row : MG132 arrested HeLa cells stably expressing tubulin - GFP ( green ) and immunostained for endogenous PRC1 ( AlexaFluor555 shown in magenta ) in 5 individual z - images . Enlargements of the boxed region ( top : merge , middle : GFP , bottom : AlexaFluor555 ) below the first image show the bridging fiber determined as the line connecting two ends of the sister k - fibers in green channel and localization of endogenous PRC1 ( magenta ) . Corresponding maximum projections are shown to the right ( max z , left : merge , middle : GFP , right : AlexaFluor555 ) . B . Quantification of tubulin - GFP bridging fibers ( green bar ) immunostained for endogenous PRC1 ( magenta bar ) .

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A. Top row: MG132 arrested HeLa cells stably expressing tubulin-GFP (green) and immunostained for endogenous PRC1 (AlexaFluor555 shown in magenta) in 5 individual z-images. Enlargements of the boxed region (top: merge, middle: GFP, bottom: AlexaFluor555) below the first image show the bridging fiber determined as the line connecting two ends of the sister k-fibers in green channel and localization of endogenous PRC1 (magenta). Corresponding maximum projections are shown to the right (max z, left: merge, middle: GFP, right: AlexaFluor555).B. Quantification of tubulin-GFP bridging fibers (green bar) immunostained for endogenous PRC1 (magenta bar).


C . Images show pole - to - pole tracking ( white curve ) of tubulin signal ( top , green ) , which was used to measure the endogenous PRC1 signal along the same contour ( middle , magenta ) , and the corresponding scheme ( bottom ) from a HeLa cell expressing tubulin - GFP and immunostained for PRC1 . Images of the same spindle without ( left ) and with the tracked contour ( right ) are shown . The graph shows normalized pole - to - pole intensity profiles ( each intensity profile was scaled so that one pole is at x = 0 and the other at x = 1 ) of endogenous PRC1 ( grey lines ) acquired in tubulin - GFP HeLa cells immunostained for PRC1 . Black line shows the mean value .

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C. Images show pole-to-pole tracking (white curve) of tubulin signal (top, green), which was used to measure the endogenous PRC1 signal along the same contour (middle, magenta), and the corresponding scheme (bottom) from a HeLa cell expressing tubulin-GFP and immunostained for PRC1. Images of the same spindle without (left) and with the tracked contour (right) are shown. The graph shows normalized pole-to-pole intensity profiles (each intensity profile was scaled so that one pole is at x=0 and the other at x=1) of endogenous PRC1 (grey lines) acquired in tubulin-GFP HeLa cells immunostained for PRC1. Black line shows the mean value.


D . Images show pole - to - pole tracking ( white curve ) of PRC1 - GFP signal ( top , merge ; middle , GFP ; bottom , scheme ) from a HeLa cell expressing PRC1 - GFP and mRFP - CENP - B . Images of the same spindle without ( left ) and with the tracked contour ( right ) are shown . The graph shows normalized pole - to - pole intensity profiles ( legend as in C ) of PRC1 - GFP acquired in HeLa cells expressing PRC1 - GFP and mRFP - CENP - B .

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D. Images show pole-to-pole tracking (white curve) of PRC1-GFP signal (top, merge; middle, GFP; bottom, scheme) from a HeLa cell expressing PRC1-GFP and mRFP-CENP-B. Images of the same spindle without (left) and with the tracked contour (right) are shown. The graph shows normalized pole-to-pole intensity profiles (legend as in C) of PRC1-GFP acquired in HeLa cells expressing PRC1-GFP and mRFP-CENP-B.


E . Example of length measurement of the immunostained PRC1 signal ( LPRC1 ) in HeLa cell expressing tubulin - GFP and immunostained for PRC1 .

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E. Example of length measurement of the immunostained PRC1 signal (LPRC1) in HeLa cell expressing tubulin-GFP and immunostained for PRC1.


F . Comparison of the length LPRC1 between PRC1 - GFP signal ( green , corresponds to the PRC1 fused to GFP from a PRC1 - GFP cell line ) and PRC1 - AlexaFluor555 ( magenta , corresponds to the endogenous immunostained PRC1 ) ( p = 0 . 10 ) .

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F. Comparison of the length LPRC1 between PRC1-GFP signal (green, corresponds to the PRC1 fused to GFP from a PRC1-GFP cell line) and PRC1-AlexaFluor555 (magenta, corresponds to the endogenous immunostained PRC1) (p=0.10).


G . Cross section signal intensity of the immunostained endogenous PRC1 ( magenta line ) from a HeLa cell expressing tubulin - GFP and immunostained for PRC1 . Area under the peak is defined as Icross , measured at the position of the blue line as in scheme . Horizontal lines mark the background signal ( black line ) , vertical lines delimit the area ( grey ) where the signal was measured .

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G. Cross section signal intensity of the immunostained endogenous PRC1 (magenta line) from a HeLa cell expressing tubulin-GFP and immunostained for PRC1. Area under the peak is defined as Icross, measured at the position of the blue line as in scheme. Horizontal lines mark the background signal (black line), vertical lines delimit the area (grey) where the signal was measured.


A . Images of HeLa cells stably expressing tubulin - GFP ( green ) and transiently mRFP - CENP - B ( magenta ) treated with control siRNA ( left images ) and siRNA targeting PRC1 ( right images ) . Enlargements of the boxed region , shown to the right of the image of the whole spindle , are focused on kinetochores and bridging fiber ( top : merge ; middle : GFP , bottom : scheme ) .

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A. Images of HeLa cells stably expressing tubulin-GFP (green) and transiently mRFP-CENP-B (magenta) treated with control siRNA (left images) and siRNA targeting PRC1 (right images). Enlargements of the boxed region, shown to the right of the image of the whole spindle, are focused on kinetochores and bridging fiber (top: merge; middle: GFP, bottom: scheme).


B . Western blot showing PRC1 protein from unlabeled cells , tubulin - GFP HeLa cell line and PRC1 - GFP HeLa cell line . Detailed measurements are in Expanded View 4 .

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B. Western blot showing PRC1 protein from unlabeled cells, tubulin-GFP HeLa cell line and PRC1-GFP HeLa cell line. Detailed measurements are in Expanded View 4.


C . Mean ratio of signal intensities of the bridging fiber ( Ib , measured at the position of a blue line as in scheme ) and sum of the bridging and k - fiber ( Ibk , measured at the position of the orange line as in scheme ) , measured in control cells ( grey bar ) and PRC1 siRNA treated cells ( black bar ) ( p = 0 . 0003 ) . D . Mean interkinetochore distance ( dk , see scheme ) measured in control cells ( grey bar ) and PRC1 siRNA treated cells ( black bar ) ( p = 0 . 0001 ) .

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C. Mean ratio of signal intensities of the bridging fiber (Ib, measured at the position of a blue line as in scheme) and sum of the bridging and k-fiber (Ibk, measured at the position of the orange line as in scheme), measured in control cells (grey bar) and PRC1 siRNA treated cells (black bar) (p=0.0003).D. Mean interkinetochore distance (dk, see scheme) measured in control cells (grey bar) and PRC1 siRNA treated cells (black bar) (p=0.0001).


E . Images of individual spindles of HeLa cells expressing tubulin - GFP and immunostained for PRC1 obtained from control cells ( top ) and PRC1 siRNA treated cells ( bottom ) . All cells were imaged with the same imaging parameters . Only AlexaFluor555 channel is shown .

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E. Images of individual spindles of HeLa cells expressing tubulin-GFP and immunostained for PRC1 obtained from control cells (top) and PRC1 siRNA treated cells (bottom). All cells were imaged with the same imaging parameters. Only AlexaFluor555 channel is shown.


F . Mean signal intensity I of the immunostained PRC1 in control cells ( grey bar ) and PRC1 siRNA treated cells ( black bar ) in HeLa cells expressing tubulin - GFP and immunostained for PRC1 ( p = 0 . 0001 ) .

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F. Mean signal intensity I of the immunostained PRC1 in control cells (grey bar) and PRC1 siRNA treated cells (black bar) in HeLa cells expressing tubulin-GFP and immunostained for PRC1 (p=0.0001).


G . Images of HeLa cells stably expressing PRC1 - GFP treated with control ( left image ) and siRNA targeting PRC1 ( right image ) immunostained for PRC1 ( magenta ) . Graph showing the length of the immunostained PRC1 signal , LPRC1 ( p = 0 . 5167 ) , signal intensity , I ( p = 0 . 0003 ) and Icross ( p = 0 . 0001 ) in siRNA targeting PRC1 in comparison to control HeLa cells expressing PRC1 - GFP and immunostained for PRC1 . H . Images of HeLa cells stably expressing PRC1 - GFP ( green ) treated with control ( left image ) and siRNA targeting PRC1 ( right image ) immunostained for PRC1 . Graph showing the length of the PRC1 - GFP signal , LPRC1 ( p = 0 . 3407 ) , signal intensity , I ( p = 0 . 0001 ) , and Icross ( p = 0 . 0001 ) in siRNA targeting PRC1 in comparison to control HeLa cells expressing PRC1 - GFP and immunostained for PRC1 .

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G. Images of HeLa cells stably expressing PRC1-GFP treated with control (left image) and siRNA targeting PRC1 (right image) immunostained for PRC1 (magenta). Graph showing the length of the immunostained PRC1 signal, LPRC1 (p=0.5167), signal intensity, I (p=0.0003) and Icross (p=0.0001) in siRNA targeting PRC1 in comparison to control HeLa cells expressing PRC1-GFP and immunostained for PRC1.H. Images of HeLa cells stably expressing PRC1-GFP (green) treated with control (left image) and siRNA targeting PRC1 (right image) immunostained for PRC1. Graph showing the length of the PRC1-GFP signal, LPRC1 (p=0.3407), signal intensity, I (p=0.0001), and Icross (p=0.0001) in siRNA targeting PRC1 in comparison to control HeLa cells expressing PRC1-GFP and immunostained for PRC1.


( A ) Proportion change of Chinese SL isolates from different phylogenetic clades . The Chinese isolates were classified into two groups based on their sampling time ( 1993 - 2010 vs 2011 - 2020 ) . The percentage of isolates from each clade in each period was compared .

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(A) Proportion change of Chinese SL isolates from different phylogenetic clades. The Chinese isolates were classified into two groups based on their sampling time (1993-2010 vs 2011-2020). The percentage of isolates from each clade in each period was compared.


( A ) Bacterial resistance to desiccation . Representative isolates that are phylogenetically distinct within Clade - 1 ( n = 2 ) , Clade - 2 ( n = 1 ) , Clade - 4 ( n = 2 ) and Clade - 5 ( n = 6 ) were used for desiccation assay Data information : All the experiments were triplicated with biological replicates . Data are presented as mean ± SD . The P values for ( A ) were calculated using unpaired t - test ( two - tailed ) ( * : P < 0 . 05 , * * : P < 0 . 01 , * * * : P < 0 . 001 ) .

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(A) Bacterial resistance to desiccation. Representative isolates that are phylogenetically distinct within Clade-1 (n=2), Clade-2 (n=1), Clade-4 (n=2) and Clade-5 (n=6) were used for desiccation assay Data information: All the experiments were triplicated with biological replicates. Data are presented as mean ± SD. The P values for (A) were calculated using unpaired t-test (two-tailed) (*:P<0.05, **:P<0.01, ***:P<0.001).


( B ) Bacterial survivability under oxidative stress . Representative isolates from C1 ( n = 2 ) , C2 ( n = 1 ) , C4 ( n = 2 ) and C5 ( n = 6 ) were treated with hydrogen peroxide . Data information : All the experiments were triplicated with biological replicates . Data are presented as mean ± SD . The P values for ( B ) , were calculated using unpaired t - test ( two - tailed ) , ( * : P < 0 . 05 , * * : P < 0 . 01 , * * * : P < 0 . 001 ) .

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(B) Bacterial survivability under oxidative stress. Representative isolates from C1 (n=2), C2 (n=1), C4 (n=2) and C5 (n=6) were treated with hydrogen peroxide. Data information: All the experiments were triplicated with biological replicates. Data are presented as mean ± SD. The P values for (B), were calculated using unpaired t-test (two-tailed), (*:P<0.05, **:P<0.01, ***:P<0.001).


( C ) Biofilm formation ability . The biofilm of the representative isolates from C1 ( n = 2 ) , C2 ( n = 1 ) , C4 ( n = 2 ) and C5 ( n = 6 ) were formed at 22 ° C for 48 h . Data information : All the experiments were triplicated with biological replicates . Data are presented as mean ± SD . The P values for ( C ) were calculated using unpaired t - test ( two - tailed ) , ( * : P < 0 . 05 , * * : P < 0 . 01 , * * * : P < 0 . 001 ) .

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(C) Biofilm formation ability. The biofilm of the representative isolates from C1 (n=2), C2 (n=1), C4 (n=2) and C5 (n=6) were formed at 22°C for 48 h. Data information: All the experiments were triplicated with biological replicates. Data are presented as mean ± SD. The P values for (C) were calculated using unpaired t-test (two-tailed), (*:P<0.05, **:P<0.01, ***:P<0.001).


( D ) Comparison of survival rates of chick embryo after challenging SL C5 and C1 isolates . Comparison was on 6 day - post - infection ( dpi ) . Ten embryos were used for each group . Data information : All the experiments were triplicated with biological replicates . Data are presented as mean ± SD . P values for ( D ) were calculated using Log - rank ( Mantel - Cox , chi - square ) test ( * : P < 0 . 05 , * * : P < 0 . 01 , * * * : P < 0 . 001 ) .

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(D) Comparison of survival rates of chick embryo after challenging SL C5 and C1 isolates. Comparison was on 6 day-post-infection (dpi). Ten embryos were used for each group. Data information: All the experiments were triplicated with biological replicates. Data are presented as mean ± SD. P values for (D) were calculated using Log-rank (Mantel-Cox, chi-square) test (*:P<0.05, **:P<0.01, ***:P<0.001).


( E ) Survival rates of zebrafish embryo after challenging SL C5 and C1 isolates . The comparison was on 14 dpi . Ten zebrafish embryos were used for each group . Data information : All the experiments were triplicated with biological replicates . Data are presented as mean ± SD . P values for ( E ) were calculated using Log - rank ( Mantel - Cox , chi - square ) test ( * : P < 0 . 05 , * * : P < 0 . 01 , * * * : P < 0 . 001 ) .

(E) Survival rates of zebrafish embryo after challenging SL C5 and C1 isolates. The comparison was on 14 dpi. Ten zebrafish embryos were used for each group. Data information: All the experiments were triplicated with biological replicates. Data are presented as mean ± SD. P values for (E) were calculated using Log-rank (Mantel-Cox, chi-square) test (*:P<0.05, **:P<0.01, ***:P<0.001).


( F ) Bacterial loads in distinct organs of mice after challenging SL C5 ( L42 ) and C1 ( L1 ) isolates . The bacterial counts in the liver , spleen , and cecum of mice were measured on 3 and 5 dpi . Ten mice were used per group . Data information : All the experiments were triplicated with biological replicates . Data are presented as mean ± SD . The P values for ( F ) were calculated using unpaired t - test ( two - tailed ) , ( * : P < 0 . 05 , * * : P < 0 . 01 , * * * : P < 0 . 001 ) .

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(F) Bacterial loads in distinct organs of mice after challenging SL C5 (L42) and C1 (L1) isolates. The bacterial counts in the liver, spleen, and cecum of mice were measured on 3 and 5 dpi. Ten mice were used per group. Data information: All the experiments were triplicated with biological replicates. Data are presented as mean ± SD. The P values for (F) were calculated using unpaired t-test (two-tailed), (*:P<0.05, **:P<0.01, ***:P<0.001).


Protein levels of E2F7 and E2F8 during cell cycle progression . HeLa cells were synchronized by a double thymidine block and released into fresh medium . Cells were harvested at the indicated time points and an asynchronous ( AS ) condition was used as control . Protein levels were measured by immunoblotting and cell cycle progression was determined by flow - cytometry ( shown in Fig EV 1A ) . The asterisk indicates the E2F7 - specific band .

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Protein levels of E2F7 and E2F8 during cell cycle progression. HeLa cells were synchronized by a double thymidine block and released into fresh medium. Cells were harvested at the indicated time points and an asynchronous (AS) condition was used as control. Protein levels were measured by immunoblotting and cell cycle progression was determined by flow-cytometry (shown in Fig EV 1A). The asterisk indicates the E2F7-specific band.


Decreased stability of E2F7 and E2F8 in nocodazole - arrested cells . HeLa cells were either treated with DMSO or nocodazole ( 50 ng / ml ) for 16 hours . Cells were harvested and lysed for immunoblotting . Protein expression of cyclin B1 was used as a marker for G2 or M , and γ - tubulin was used as loading control .

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Decreased stability of E2F7 and E2F8 in nocodazole-arrested cells. HeLa cells were either treated with DMSO or nocodazole (50 ng/ml) for 16 hours. Cells were harvested and lysed for immunoblotting. Protein expression of cyclin B1 was used as a marker for G2 or M, and γ-tubulin was used as loading control.


Selective Cullin - RING inhibitor MLN4924 rescued the degradation of E2F7 / 8 under nocodazole - arrested condition . HeLa cells were treated with DMSO , nocodazole or nocodazole plus MLN4924 ( 0 . 1 μM ) for 16 hours . Cells were harvested and lysed for immunoblotting . cyclin B1 expression was used as a marker for G2 or M cell cycle progression , and γ - tubulin was used as loading control .

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Selective Cullin-RING inhibitor MLN4924 rescued the degradation of E2F7/8 under nocodazole-arrested condition. HeLa cells were treated with DMSO, nocodazole or nocodazole plus MLN4924 (0.1 μM) for 16 hours. Cells were harvested and lysed for immunoblotting. cyclin B1 expression was used as a marker for G2 or M cell cycle progression, and γ-tubulin was used as loading control.


Increased half - life of E2F7 / 8 by MLN4924 treatment . HeLa cells were either treated with cycloheximide ( CHX , 50 ug / ml ) with or without MLN4924 ( 0 . 1 μM ) . Protein levels of E2F7 and E2F8 were determined by immunoblotting ( left panel ) . Asterisk indicates the E2F7 - specific band . cyclin B1 and cyclin A2 expressions were used as a marker for G2 or M cell cycle progression , γ - tubulin was used as loading control . Quantifications ( right panels ) were performed based on two independent experiments . Bar and error bars represent mean ± SEM .

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Increased half-life of E2F7/8 by MLN4924 treatment. HeLa cells were either treated with cycloheximide (CHX, 50 ug/ml) with or without MLN4924 (0.1 μM). Protein levels of E2F7 and E2F8 were determined by immunoblotting (left panel). Asterisk indicates the E2F7-specific band. cyclin B1 and cyclin A2 expressions were used as a marker for G2 or M cell cycle progression, γ-tubulin was used as loading control. Quantifications (right panels) were performed based on two independent experiments. Bar and error bars represent mean ± SEM.


C - terminus motifs at parallel positions on E2F7 and E2F8 are essential for binding to cyclin F . The residues on each motif were mutated to alanines ( R to A , I / L to A ) with site - direct mutagenesis PCR . HEK293 cells were transfected with either EGFP - tagged empty vector ( EGFP ) , wild - types E2F7 / 8 ( WT ) or alanine mutants . Nocodazole ( 50 ng / ml ) was added 32 hours after transfection , and MG132 ( 1 μg / ml ) was added 5 hours before harvesting at 48 hours post transfection . Cells were harvested and lysed for immunoprecipitation using anti - EGFP resin followed by immunoblotting with antibodies against cyclin F and EGFP .

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C-terminus motifs at parallel positions on E2F7 and E2F8 are essential for binding to cyclin F. The residues on each motif were mutated to alanines (R to A, I/L to A) with site-direct mutagenesis PCR. HEK293 cells were transfected with either EGFP-tagged empty vector (EGFP), wild-types E2F7/8 (WT) or alanine mutants. Nocodazole (50 ng/ml) was added 32 hours after transfection, and MG132 (1 μg/ml) was added 5 hours before harvesting at 48 hours post transfection. Cells were harvested and lysed for immunoprecipitation using anti-EGFP resin followed by immunoblotting with antibodies against cyclin F and EGFP.


Schematic view showing the truncated mutants and ML / AA mutant of cyclin F ( Left ) . HEK cells were transfected with indicated constructs and co - immunoprecipitation was performed using Flag resin ( Right ) .

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Schematic view showing the truncated mutants and ML/AA mutant of cyclin F (Left). HEK cells were transfected with indicated constructs and co-immunoprecipitation was performed using Flag resin (Right).


Wild - type or mutant versions of EGFP - tagged E2F7 / 8 were co - transfected with either empty vector or Flag - tagged cyclin F in HEK293 cells . Nocodazole was added to cells 8h before harvest . 48 hours after transfection , cells were collected and lysed for immunoblotting .

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Wild-type or mutant versions of EGFP-tagged E2F7/8 were co-transfected with either empty vector or Flag-tagged cyclin F in HEK293 cells. Nocodazole was added to cells 8h before harvest. 48 hours after transfection, cells were collected and lysed for immunoblotting.


Knockdown of cyclin F stabilized E2F7 and E2F8 . HeLa and RPE cells were transfected with either scramble siRNA or pool cyclin F siRNA . Cells were harvested at 48 hours post transfection . Protein levels of E2F7 / 8 were analyzed by immunoblotting . Asterisk indicates the specific band of E2F7 detection .

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Knockdown of cyclin F stabilized E2F7 and E2F8. HeLa and RPE cells were transfected with either scramble siRNA or pool cyclin F siRNA. Cells were harvested at 48 hours post transfection. Protein levels of E2F7/8 were analyzed by immunoblotting. Asterisk indicates the specific band of E2F7 detection.


Cyclin F targets atypical E2Fs during G2 / M . HeLa cells were transfected with either scrambled siRNA ( scr ) or cyclin F siRNA ( sicyclin F ) for 24 hours . Then cells were synchronized by double thymidine block and released into fresh medium after the second block . Cells were harvested at the indicated timepoints after the release . Protein expression was measured by immunoblotting and cell cycle progression was determined by flow - cytometry ( shown in Fig EV 1A ) . Asterisk indicates the specific detection of endogenous E2F7 .

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Cyclin F targets atypical E2Fs during G2/M. HeLa cells were transfected with either scrambled siRNA (scr) or cyclin F siRNA (sicyclin F) for 24 hours. Then cells were synchronized by double thymidine block and released into fresh medium after the second block. Cells were harvested at the indicated timepoints after the release. Protein expression was measured by immunoblotting and cell cycle progression was determined by flow-cytometry (shown in Fig EV 1A). Asterisk indicates the specific detection of endogenous E2F7.


Cyclin F contributes to the ubiquitylation of E2F7 and E2F8 in vivo . HEK293 cells were transfected with HA - E2F7 / 8 , with or without Flag - cyclin F , and with HA - tagged ubiquitin . 5 hours before harvest , cells were treated with MG132 . 48 hours after transfection , HEK cells were harvested and lysed for immunoprecipitation pull - down assay with anti - HA resin followed by immunoblotting .

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Cyclin F contributes to the ubiquitylation of E2F7 and E2F8 in vivo. HEK293 cells were transfected with HA-E2F7/8, with or without Flag-cyclin F, and with HA-tagged ubiquitin. 5 hours before harvest, cells were treated with MG132. 48 hours after transfection, HEK cells were harvested and lysed for immunoprecipitation pull-down assay with anti-HA resin followed by immunoblotting.


Schematic view of the experimental setting for the HU - synchronized live cell imaging . 48 hours before imaging , RPE - FUCCI cells were transfected with siRNA against scramble or cyclin F . 16 hours before imaging , cells were synchronized at the G1 / S border by HU ( 2 mM ) treatment . Representative images from different channels are shown , and white arrows in Differential Interference Contrast DIC channel indicate the traced cell . Scale bar : 10µm .

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Schematic view of the experimental setting for the HU-synchronized live cell imaging. 48 hours before imaging, RPE-FUCCI cells were transfected with siRNA against scramble or cyclin F. 16 hours before imaging, cells were synchronized at the G1/S border by HU (2 mM) treatment. Representative images from different channels are shown, and white arrows in Differential Interference Contrast DIC channel indicate the traced cell. Scale bar: 10µm.


Quantification of the number of Ctrl ( left panel ) and 7 / 8KO ( right panel ) RPE - FUCCI cells with scr or sicyclin F that completed mitosis after HU release . For each condition , 100 cells were monitored by live cell imaging . Each cell was followed until it successfully finished mitosis and divided into two daughter cells for a maximum of 24 hours . Log - rank tests were performed to analyze the statistical significance .

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Quantification of the number of Ctrl (left panel) and 7/8KO (right panel) RPE-FUCCI cells with scr or sicyclin F that completed mitosis after HU release. For each condition, 100 cells were monitored by live cell imaging. Each cell was followed until it successfully finished mitosis and divided into two daughter cells for a maximum of 24 hours. Log-rank tests were performed to analyze the statistical significance.


Schematic view of the experimental setting for live imaging of asynchronous cells . 48 hours before imaging , RPE - FUCCI cells were transfected with siRNA against scramble or cyclin F . At the start of the imaging , G1 cells ( red : mKO2 - Cdt1 > mAG1 - Geminin , 50 cells per condition ) were enrolled and subsequently monitored though their entire cell cycle until mitosis . The black dot represents mitosis .

Schematic view of the experimental setting for live imaging of asynchronous cells. 48 hours before imaging, RPE-FUCCI cells were transfected with siRNA against scramble or cyclin F. At the start of the imaging, G1 cells (red: mKO2-Cdt1 > mAG1-Geminin, 50 cells per condition) were enrolled and subsequently monitored though their entire cell cycle until mitosis. The black dot represents mitosis.


Knockdown of cyclin F causes a delay in mitotic entry that is dependent on E2F7 and E2F8 . Number of Ctrl ( left panel ) or 7 / 8KO ( right panel ) RPE - FUCCI cells with scr or cyclin F RNAi that finished mitosis during live cell imaging is shown . For each condition , 50 cells at G1 were monitored by live cell imaging . Each cell was followed until it successfully progressed through S and G2 phase , finished mitosis and divided into two daughter cells , for a maximum of 24 hours . Log - rank tests were performed to analyze the statistical significance .

Knockdown of cyclin F causes a delay in mitotic entry that is dependent on E2F7 and E2F8. Number of Ctrl (left panel) or 7/8KO (right panel) RPE-FUCCI cells with scr or cyclin F RNAi that finished mitosis during live cell imaging is shown. For each condition, 50 cells at G1 were monitored by live cell imaging. Each cell was followed until it successfully progressed through S and G2 phase, finished mitosis and divided into two daughter cells, for a maximum of 24 hours. Log-rank tests were performed to analyze the statistical significance.


Loss of cyclin F delays the progression from G1 / S transition to mitosis . Histogram shows the time from G1 / S ( mAG1 - Geminin intensity increases to higher than 10 % of the maximum value in three consecutive imaging frames ) to completed mitosis . Only cells that finished mitosis were enrolled in this quantification . Student t - test was used to test the statistical significance , asterisks indicates the P value < 0 . 01 . Bar and error bars represent mean ± SEM .

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Loss of cyclin F delays the progression from G1/S transition to mitosis. Histogram shows the time from G1/S (mAG1-Geminin intensity increases to higher than 10% of the maximum value in three consecutive imaging frames) to completed mitosis. Only cells that finished mitosis were enrolled in this quantification. Student t-test was used to test the statistical significance, asterisks indicates the P value <0.01. Bar and error bars represent mean ± SEM.


Depletion of cyclin F stalls the cell cycle at late S / G2 . After 24 hours of live cell imaging , the cell cycle progression from panel E was quantified . Histogram shows the percentage of cells at each stage ( at 24h ) over the whole population ( 50 cells per condition ) .

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Depletion of cyclin F stalls the cell cycle at late S/G2. After 24 hours of live cell imaging, the cell cycle progression from panel E was quantified. Histogram shows the percentage of cells at each stage (at 24h) over the whole population (50 cells per condition).


Disruption of cyclin F binding site increased stability of E2F7 . E2F7WT and E2F7R894A constructs were integrated in to HeLa / TO system . Cells were arrested with HU for 16 hours before releasing into doxycycline containing medium , and cells were collected every 3 hours for immunoblotting .

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Disruption of cyclin F binding site increased stability of E2F7. E2F7WT and E2F7R894A constructs were integrated in to HeLa/TO system. Cells were arrested with HU for 16 hours before releasing into doxycycline containing medium, and cells were collected every 3 hours for immunoblotting.


qPCR showing that mRNA level of E2F7WT and E2F7R894A were at comparable levels . Bar and error bars represent mean ± SEM .

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qPCR showing that mRNA level of E2F7WT and E2F7R894A were at comparable levels. Bar and error bars represent mean ± SEM.


Over - expression of E2F7R894A delays cell cycle progression through G2 - M phase . HeLa / TO cells expressing either wild - type or mutant version of E2F7 were arrested with 16h of HU , and then cells were released into fresh medium with or without doxycycline . Live cell imaging was performed to trace the G2 - M progression of HeLa / TO cells .

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Over-expression of E2F7R894A delays cell cycle progression through G2-M phase. HeLa/TO cells expressing either wild-type or mutant version of E2F7 were arrested with 16h of HU, and then cells were released into fresh medium with or without doxycycline. Live cell imaging was performed to trace the G2-M progression of HeLa/TO cells.


Heatmap showing differentially expressed genes after cyclin F knockdown , and rescued by additional E2F7 / 8 depletion . Highlighted genes are all involved in DNA repair . Cells were arrested with nocodazole for 16 hours prior to harvesting to minimize bias from potential differences in cell cycle progression between the different conditions .

Heatmap showing differentially expressed genes after cyclin F knockdown, and rescued by additional E2F7/8 depletion. Highlighted genes are all involved in DNA repair. Cells were arrested with nocodazole for 16 hours prior to harvesting to minimize bias from potential differences in cell cycle progression between the different conditions.


qPCR showing the RNA expression of atypical E2F - target genes that are involved in DNA replication or DNA repair . HeLa cells were transfected for 48 hours with siRNAs as indicated . Cells were incubated with nocodazole for 16 hours before harvesting . Data represent averages ± SEM ( n = 3 ) ; * P < 0 . 05 or * * P < 0 . 01 ( Student ' s t - test ) . n . s . : not significant .

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qPCR showing the RNA expression of atypical E2F- target genes that are involved in DNA replication or DNA repair. HeLa cells were transfected for 48 hours with siRNAs as indicated. Cells were incubated with nocodazole for 16 hours before harvesting. Data represent averages ± SEM (n=3); *P<0.05 or **P<0.01 (Student's t-test). n.s.: not significant.


Immunoblotting showing the protein levels of Chk1 and Rad51 in the indicated siRNA conditions . HeLa cells were treated with nocodazole for 16 hours prior to harvesting .

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Immunoblotting showing the protein levels of Chk1 and Rad51 in the indicated siRNA conditions. HeLa cells were treated with nocodazole for 16 hours prior to harvesting.


Loss of cyclin F induced E2F7 / 8 - dependent Homologous Recombination deficiency . HeLa cells that were stably transformed with pDR - GFP were transfected with siRNA as indicated . 48h after the initial transfection , cells were harvested for Flow - cytometry . GFP positive cells were gated ( Fig EV5A ) . Relative HR efficiency were adjusted to the scramble siRNA condition . Data represent averages ± SEM ( n = 3 ) ; * * P < 0 . 01 ( Student ' s t - test ) . n . s . : not significant .

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Loss of cyclin F induced E2F7/8-dependent Homologous Recombination deficiency. HeLa cells that were stably transformed with pDR-GFP were transfected with siRNA as indicated.48h after the initial transfection, cells were harvested for Flow-cytometry. GFP positive cells were gated (Fig EV5A). Relative HR efficiency were adjusted to the scramble siRNA condition. Data represent averages ± SEM (n=3); **P<0.01 (Student's t-test). n.s.: not significant.


Loss of cyclin F induced E2F7 / 8 - dependent γ - H2AX accumulation . HeLa cells were transfected with indicated siRNA for 24 hours , and then treated with nocodazole for 16 hours before fixation for immunofluorescence staining of γ - H2AX . DAPI was used to stain the cell nucleus . Relative intensity of γ - H2AX was quantified by Image J software , and 150 cells were quantified for each condition . Red bars represent averages ; * * P < 0 . 01 ( Student ' s t - test ) . n . s . : not significant . Scale bar 20 μm .

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Loss of cyclin F induced E2F7/8-dependent γ-H2AX accumulation. HeLa cells were transfected with indicated siRNA for 24 hours, and then treated with nocodazole for 16 hours before fixation for immunofluorescence staining of γ-H2AX. DAPI was used to stain the cell nucleus. Relative intensity of γ-H2AX was quantified by Image J software, and 150 cells were quantified for each condition. Red bars represent averages; **P<0.01 (Student's t-test). n.s.: not significant. Scale bar 20 μm.


Loss of cyclin F increased DNA damage recovery time before cell division . RPE cells integrated with the 53BP1 construct were transfected with indicated siRNA for 24 hours , and then treated with HU for 16 hours . At the beginning of the imaging , only the single cells with at least one 53BP1 foci were traced , till the time frame that no 53BP1 foci was observed . The mitotic progression of the cells was defined as the duration from damage recovery to cell division . Histogram shows the damage recovery time ( green ) and the mitotic progression ( black ) of 50 cells for each condition . Chi - square analysis was performed to test the statistical significance ( P < 0 . 01 ) .

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Loss of cyclin F increased DNA damage recovery time before cell division. RPE cells integrated with the 53BP1 construct were transfected with indicated siRNA for 24 hours, and then treated with HU for 16 hours. At the beginning of the imaging, only the single cells with at least one 53BP1 foci were traced, till the time frame that no 53BP1 foci was observed. The mitotic progression of the cells was defined as the duration from damage recovery to cell division. Histogram shows the damage recovery time (green) and the mitotic progression (black) of 50 cells for each condition. Chi-square analysis was performed to test the statistical significance (P<0.01).


Knockdown of cyclin F caused a delay in DNA lesion recovery that is dependent on E2F7 / 8 . The cumulative curves represent the add - up number of cells that overcome the DNA damage lesions , for a time frame of 24 hours . For each condition , 50 cells were quantified . Log - rank tests were performed to analyze the statistical significance .

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Knockdown of cyclin F caused a delay in DNA lesion recovery that is dependent on E2F7/8. The cumulative curves represent the add-up number of cells that overcome the DNA damage lesions, for a time frame of 24 hours. For each condition, 50 cells were quantified. Log-rank tests were performed to analyze the statistical significance.


ALP assay . The cells expressing Pho8Δ60p were cultured in SD + CA medium ( open bars ) and shifted to SD ( − N ) medium for three hours ( filled bars ) . The ALP activity of the lysates was measured to estimate autophagic activity . Wild - type cells , TN124 ; Δapg8 mutant harboring vector , TK303 ; Δapg8 mutant harboring APG8 on a centromeric vector , TK301 ; Δapg8 mutant harboring 3 × HA - tagged APG8 plasmid , TK307 .

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ALP assay. The cells expressing Pho8Δ60p were cultured in SD+CA medium (open bars) and shifted to SD(−N) medium for three hours (filled bars). The ALP activity of the lysates was measured to estimate autophagic activity. Wild-type cells, TN124; Δapg8 mutant harboring vector, TK303; Δapg8 mutant harboring APG8 on a centromeric vector, TK301; Δapg8 mutant harboring 3 × HA-tagged APG8 plasmid, TK307.


Expression of APG8 . A , The lysates prepared from the growing cells were subjected to immunoblotting with the anti - Apg8p antibody . Lane 1 , wild - type cells ( YW5 - 1B ) ; lane 2 , Δapg8 cells harboring APG8 on a multicopy vector ( TK201 ) ; lane 3 , Δapg8 cells ( TK404 ) .

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Expression of APG8. A, The lysates prepared from the growing cells were subjected to immunoblotting with the anti-Apg8p antibody. Lane 1, wild-type cells (YW5-1B); lane 2, Δapg8 cells harboring APG8 on a multicopy vector (TK201); lane 3, Δapg8 cells (TK404).


B , The wild - type ( YW5 - 1B ) cells were cultured in YEPD medium until 1 - 2 × 107 cells / ml ( 0 h ) and shifted to SD ( − N ) medium for 0 . 5 , 1 , 2 , 3 , 4 . 5 , and 6 h at 30 ° C . The amount of Apg8p was estimated by immunoblotting with anti - Apg8p antibody . Each lane has 10 μg of total protein .

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B, The wild-type (YW5-1B) cells were cultured in YEPD medium until 1-2 × 107 cells/ml (0 h) and shifted to SD(−N) medium for 0.5, 1, 2, 3, 4.5, and 6 h at 30°C. The amount of Apg8p was estimated by immunoblotting with anti-Apg8p antibody. Each lane has 10 μg of total protein.


C , YW5 - 1B cells were cultured in YEPD medium until 1 - 2 × 107 cells / ml ( 0 h ) and shifted to SD ( − N ) medium for 0 . 5 , 1 , 2 , and 6 h at 30 ° C , and mRNA was prepared from each culture as described in Materials and Methods . APG8 mRNA and ACT1 mRNA were detected by Northern blotting with each specific probe . Each lane has 4 μg of total mRNA .

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C, YW5-1B cells were cultured in YEPD medium until 1-2 × 107 cells/ml (0 h) and shifted to SD(−N) medium for 0.5, 1, 2, and 6 h at 30°C, and mRNA was prepared from each culture as described in Materials and Methods. APG8 mRNA and ACT1 mRNA were detected by Northern blotting with each specific probe. Each lane has 4 μg of total mRNA.


D , YW5 - 1B cells were cultured in YEPD medium until 1 × 107 cells / ml at 30 ° C . Rapamycin was added to the culture at a final concentration of 0 . 2 μg / ml and the cells were further incubated in YEPD medium for two hours at 30 ° C . Western blotting was performed with the anti - Apg8p antibody . Each lane has 20 μg of total protein .

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D, YW5-1B cells were cultured in YEPD medium until 1 × 107 cells/ml at 30°C. Rapamycin was added to the culture at a final concentration of 0.2 μg/ml and the cells were further incubated in YEPD medium for two hours at 30°C. Western blotting was performed with the anti-Apg8p antibody. Each lane has 20 μg of total protein.


Subcellular fractionation and solubilization of Apg8p . The wild - type cells ( YW5 - 1B ) growing logarithmically in YEPD medium or starved in SD ( − N ) medium for three hours were lysed . A , The lysates ( T ) were centrifuged at 13 , 000 g for 15 min to generate pellet ( LSP ) and supernatant ( LSS ) . LSS fraction was further centrifuged at 100 , 000 g for one hour and separated to pellet ( HSP ) and supernatant ( HSS ) . LSP and HSP were resuspended in an equal volume of the lysis buffer to the original lysates . Equal volume of each sample was applied to each well , and immunoblotting was performed with anti - Apg8p antibody .

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Subcellular fractionation and solubilization of Apg8p. The wild-type cells (YW5-1B) growing logarithmically in YEPD medium or starved in SD(−N) medium for three hours were lysed. A, The lysates (T) were centrifuged at 13,000 g for 15 min to generate pellet (LSP) and supernatant (LSS). LSS fraction was further centrifuged at 100,000 g for one hour and separated to pellet (HSP) and supernatant (HSS). LSP and HSP were resuspended in an equal volume of the lysis buffer to the original lysates. Equal volume of each sample was applied to each well, and immunoblotting was performed with anti-Apg8p antibody.