Left : NIH - 3T3 cells expressing sgCtrl , sgP5CS - 1 or sgP5CS - 2 were treated with TGFβ or mock for 48h , and proline abundance was measured by GC - MS . Values are relative to mock - treated sgCtrl expressing cells . Right : western blot of NIH - 3T3 cells expressing sgCtrl , sgP5CS - 1 or sgP5CS - 2 .

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

Left: NIH-3T3 cells expressing sgCtrl, sgP5CS-1 or sgP5CS-2 were treated with TGFβ or mock for 48h, and proline abundance was measured by GC-MS. Values are relative to mock-treated sgCtrl expressing cells. Right: western blot of NIH-3T3 cells expressing sgCtrl, sgP5CS-1 or sgP5CS-2.


Western blot of NIH - 3T3 cells expressing sgCtrl or sgP5CS - 2 and treated with TGFβ or mock for 48h in the presence or absence of 0 . 15 mM proline .

O O O O O O O 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

Western blot of NIH-3T3 cells expressing sgCtrl or sgP5CS-2 and treated with TGFβ or mock for 48h in the presence or absence of 0.15 mM proline.


Collagen abundance in ECM produced by NIH - 3T3 cells expressing sgCtrl or sgP5CS - 2 grown in the presence or absence of TGFβ and 0 . 15 mM proline , measured by picrosirius red staining and normalized to the packed cell volume of cells grown on a parallel plate under identical conditions . Values are relative to mock - treated sgCtrl expressing cells .

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

Collagen abundance in ECM produced by NIH-3T3 cells expressing sgCtrl or sgP5CS-2 grown in the presence or absence of TGFβ and 0.15 mM proline, measured by picrosirius red staining and normalized to the packed cell volume of cells grown on a parallel plate under identical conditions. Values are relative to mock-treated sgCtrl expressing cells.


Proline abundance in NIH - 3T3 cells expressing empty vector or HA - P5CS cDNA , measured by GC - MS . Values are relative to mock - treated empty vector expressing cells .

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

Proline abundance in NIH-3T3 cells expressing empty vector or HA-P5CS cDNA, measured by GC-MS. Values are relative to mock-treated empty vector expressing cells.


Western blot of NIH - 3T3 cells expressing empty vector or HA - P5CS cDNA .

Western blot of NIH-3T3 cells expressing empty vector or HA-P5CS cDNA.


Collagen abundance in ECM produced by NIH - 3T3 cells expressing empty vector or HA - P5CS cDNA , measured by picrosirius red staining and normalized to the packed cell volume of cells grown on a parallel plate under identical conditions . Values are relative to mock -

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

Collagen abundance in ECM produced by NIH-3T3 cells expressing empty vector or HA-P5CS cDNA, measured by picrosirius red staining and normalized to the packed cell volume of cells grown on a parallel plate under identical conditions. Values are relative to mock-treated


Analysis of the indicated gene expression datasets for mRNA levels of P5CS : ( i ) lung tissue from mice with pulmonary fibrosis induced by bleomycin ( Bleo ) treatment compared to saline treatment ( GSE112827 ) ;

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

Analysis of the indicated gene expression datasets for mRNA levels of P5CS: (i) lung tissue from mice with pulmonary fibrosis induced by bleomycin (Bleo) treatment compared to saline treatment (GSE112827);


two datasets ( GSE110147 , GSE32537 ) from lungs of patients with idiopathic pulmonary fibrosis ( IPF ) compared to normal controls ( Ctrl ) . AU , arbitrary units . The number of patients per group is indicated .

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

two datasets (GSE110147, GSE32537) from lungs of patients with idiopathic pulmonary fibrosis (IPF) compared to normal controls (Ctrl). AU, arbitrary units. The number of patients per group is indicated.


Pearson correlation of P5CS mRNA level and forced vital capacity ( FVC ) before bronchodilator ( pre - BD ) as percentage of what was predicted for each patient , from clinical data of GSE32537 .

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

Pearson correlation of P5CS mRNA level and forced vital capacity (FVC) before bronchodilator (pre-BD) as percentage of what was predicted for each patient, from clinical data of GSE32537.


Tracing of [ U - 13C ] L - glutamine ( [ U - 13C ] Gln ) into indicated metabolites . NIH - 3T3 cells expressing sgCtrl , sgSmad4 - 1 or sgSmad4 - 2 were treated with TGFβ or mock for 48h and the medium was replaced ( including all treatments ) with DMEM without L - glutamine and supplemented with [ U - 13C ] Gln for the last 8h . Metabolites were measured by GC - MS .

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

Tracing of [U-13C] L-glutamine ([U-13C] Gln) into indicated metabolites. NIH-3T3 cells expressing sgCtrl, sgSmad4-1 or sgSmad4-2 were treated with TGFβ or mock for 48h and the medium was replaced (including all treatments) with DMEM without L-glutamine and supplemented with [U-13C] Gln for the last 8h. Metabolites were measured by GC-MS.


Mitochondrial basal respiration ( a ) and FCCP - induced maximal respiration ( b ) of NIH - 3T3 cells treated with TGFβ ( 2 ng / mL for all experiments ) or mock for 2h , 4h , 8h , 24h or 48h , based on Seahorse OCR measurements . The relative values were calculated by normalizing the respective values of TGFβ - treated cells to those of mock - treated cells for each time point .

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

Mitochondrial basal respiration (a) and FCCP-induced maximal respiration (b) of NIH-3T3 cells treated with TGFβ (2 ng/mL for all experiments) or mock for 2h, 4h, 8h, 24h or 48h, based on Seahorse OCR measurements. The relative values were calculated by normalizing the respective values of TGFβ-treated cells to those of mock-treated cells for each time point.


Left : western blot of NIH - 3T3 cells expressing empty vector , mitoTPNOX or mitoLbNOX . MitoTPNOX or mitoLbNOX expression is detected by probing for their FLAG - tag . Right : NIH - 3T3 cells expressing empty vector , mitoTPNOX or mitoLbNOX were treated with TGFβ or mock for 48h and proline abundance was measured by GC - MS . Values are normalized to mock - treated empty vector expressing cells .

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

Left: western blot of NIH-3T3 cells expressing empty vector, mitoTPNOX or mitoLbNOX. MitoTPNOX or mitoLbNOX expression is detected by probing for their FLAG-tag. Right: NIH-3T3 cells expressing empty vector, mitoTPNOX or mitoLbNOX were treated with TGFβ or mock for 48h and proline abundance was measured by GC-MS. Values are normalized to mock-treated empty vector expressing cells.


NIH - 3T3 cells were treated with rotenone , antimycin , myxothiazol or vehicle control for 1h , and proline abundance was measured by GC - MS . Values are normalized to control - treated cells .

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

NIH-3T3 cells were treated with rotenone, antimycin, myxothiazol or vehicle control for 1h, and proline abundance was measured by GC-MS. Values are normalized to control-treated cells.


NIH - 3T3 cells were treated with cobalt chloride ( CoCl2 ) or vehicle control for 6h , and proline abundance was measured by GC - MS . Values are normalized to control - treated cells .

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

NIH-3T3 cells were treated with cobalt chloride (CoCl2) or vehicle control for 6h, and proline abundance was measured by GC-MS. Values are normalized to control-treated cells.


NIH - 3T3 cells were treated with TGFβ or mock for 48h , and FCCP or vehicle control was added for the last 6h of the treatment . Proline abundance was measured by GC - MS . Values are normalized to mock - and control - treated cells .

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

NIH-3T3 cells were treated with TGFβ or mock for 48h, and FCCP or vehicle control was added for the last 6h of the treatment. Proline abundance was measured by GC-MS. Values are normalized to mock- and control-treated cells.


NIH - 3T3 cells were treated with TGFβ or mock for 48h , and oligomycin or vehicle control was added for the last 1h or 6h of the treatment . Proline abundance was measured by GC - MS . Values are normalized to mock - and control - treated cells .

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

NIH-3T3 cells were treated with TGFβ or mock for 48h, and oligomycin or vehicle control was added for the last 1h or 6h of the treatment. Proline abundance was measured by GC-MS. Values are normalized to mock- and control-treated cells.


NIH - 3T3 cells were treated with TGFβ or mock for 1h , 6h , 24h or 48h . ROS levels were measured by flow cytometry for CM - H2DCFDA after incubation with CM - H2DCFDA for the last 30 min of the treatment ( Red line ) . Proline abundance was measured by GC - MS ( Blue line ) . The relative levels were calculated by normalizing the respective values of TGFβ - treated cells to those of mock - treated cells for each time point .

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

NIH-3T3 cells were treated with TGFβ or mock for 1h, 6h, 24h or 48h. ROS levels were measured by flow cytometry for CM-H2DCFDA after incubation with CM-H2DCFDA for the last 30 min of the treatment (Red line). Proline abundance was measured by GC-MS (Blue line). The relative levels were calculated by normalizing the respective values of TGFβ-treated cells to those of mock-treated cells for each time point.


Western blot of NIH - 3T3 cells were treated with TGFβ or mock for 1h , 6h , 24h or 48h .

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

Western blot of NIH-3T3 cells were treated with TGFβ or mock for 1h, 6h, 24h or 48h.


NIH - 3T3 cells expressing sgCtrl , sgSmad4 - 1 or sgSmad4 - 2 were treated with TGFβ or mock for 6h , and ROS levels were measured by flow cytometry for CM - H2DCFDA after incubation with CM - H2DCFDA for the last 30 min of the treatment . Values are normalized to mock - treated sgCtrl expressing cells .

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

NIH-3T3 cells expressing sgCtrl, sgSmad4-1 or sgSmad4-2 were treated with TGFβ or mock for 6h, and ROS levels were measured by flow cytometry for CM-H2DCFDA after incubation with CM-H2DCFDA for the last 30 min of the treatment. Values are normalized to mock-treated sgCtrl expressing cells.


Western blot of NIH - 3T3 cells expressing sgCtrl , sgSmad4 - 1 or sgSmad4 - 2 and treated with TGFβ or mock for 6h in the presence of 0 . 5 % FBS .

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

Western blot of NIH-3T3 cells expressing sgCtrl, sgSmad4-1 or sgSmad4-2 and treated with TGFβ or mock for 6h in the presence of 0.5% FBS.


NIH - 3T3 cells expressing mito - Grx1 - roGFP2 or cyto - Grx1 - roGFP2 were treated with TGFβ or mock for 6h or with 100 µM H2O2 as a control . Emission was measured with a 520 / 10nm filter after excitation with 405nm and 488nm lasers using flow cytometry . Oxidized roGFP2 gains excitability at 405nm and loses excitability at 488nm . Oxidation status is expressed as percentage of maximal oxidation which was determined by treating cells with 5 mM H2O2 for 5 min before harvest .

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

NIH-3T3 cells expressing mito-Grx1-roGFP2 or cyto-Grx1-roGFP2 were treated with TGFβ or mock for 6h or with 100 µM H2O2 as a control. Emission was measured with a 520/10nm filter after excitation with 405nm and 488nm lasers using flow cytometry. Oxidized roGFP2 gains excitability at 405nm and loses excitability at 488nm. Oxidation status is expressed as percentage of maximal oxidation which was determined by treating cells with 5 mM H2O2 for 5 min before harvest.


NIH - 3T3 cells expressing sgCtrl or sgP5CS - 2 were treated with TGFβ or mock for 48h in the presence of 0 . 15 mM proline . ROS levels were measured by flow cytometry for CM - H2DCFDA after incubation with CM - H2DCFDA for the last 30 min of the treatment .

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

NIH-3T3 cells expressing sgCtrl or sgP5CS-2 were treated with TGFβ or mock for 48h in the presence of 0.15 mM proline. ROS levels were measured by flow cytometry for CM-H2DCFDA after incubation with CM-H2DCFDA for the last 30 min of the treatment.


NIH - 3T3 cells were treated with vehicle control or CTPi at increasing concentrations ( 0 . 1 mM , 0 . 2 mM , 0 . 5 mM ) or TGFβ ( 2 ng / mL for all experiments ) for 6h , and ROS levels were measured by flow cytometry for CM - H2DCFDA after incubation with CM - H2DCFDA for the last 30 min of the treatment . Values are relative to control - treated cells . P - values represent comparison of individual samples to the control .

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

NIH-3T3 cells were treated with vehicle control or CTPi at increasing concentrations (0.1 mM, 0.2 mM, 0.5 mM) or TGFβ (2 ng/mL for all experiments) for 6h, and ROS levels were measured by flow cytometry for CM-H2DCFDA after incubation with CM-H2DCFDA for the last 30 min of the treatment. Values are relative to control-treated cells. P-values represent comparison of individual samples to the control.


Left : NIH - 3T3 cells expressing sgCtrl , sgSlc25a1 - 1 or sgSlc25a1 - 2 were treated with TGFβ or mock for 6h , and ROS levels were measured by flow cytometry for CM - H2DCFDA after incubation with CM - H2DCFDA for the last 30 min of the treatment . Values are relative to mock - treated sgCtrl expressing cells . Right : western blot of NIH - 3T3 cells expressing sgCtrl , sgSlc25a1 - 1 or sgSlc25a1 - 2 .

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

Left: NIH-3T3 cells expressing sgCtrl, sgSlc25a1-1 or sgSlc25a1-2 were treated with TGFβ or mock for 6h, and ROS levels were measured by flow cytometry for CM-H2DCFDA after incubation with CM-H2DCFDA for the last 30 min of the treatment. Values are relative to mock-treated sgCtrl expressing cells. Right: western blot of NIH-3T3 cells expressing sgCtrl, sgSlc25a1-1 or sgSlc25a1-2.


NIH - 3T3 cells expressing sgCtrl , sgSlc25a1 - 1 or sgSlc25a1 - 2 were treated with TGFβ or mock for 48h and proline abundance was measured by GC - MS . Values are relative to mock - treated sgCtrl expressing cells .

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

NIH-3T3 cells expressing sgCtrl, sgSlc25a1-1 or sgSlc25a1-2 were treated with TGFβ or mock for 48h and proline abundance was measured by GC-MS. Values are relative to mock-treated sgCtrl expressing cells.


Western blot of NIH - 3T3 cells expressing sgCtrl , sgSlc25a1 - 1 or sgSlc25a1 - 2 and treated with TGFβ or mock for 48h .

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

Western blot of NIH-3T3 cells expressing sgCtrl, sgSlc25a1-1 or sgSlc25a1-2 and treated with TGFβ or mock for 48h.


Collagen abundance in ECM produced by NIH - 3T3 cells expressing sgCtrl , sgSlc25a1 - 1 or sgSlc25a1 - 2 grown in the presence or absence of TGFβ , measured by picrosirius red staining and normalized to the packed cell volume of cells grown on a parallel plate under identical conditions . Values are relative to mock - treated sgCtrl expressing cells .

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

Collagen abundance in ECM produced by NIH-3T3 cells expressing sgCtrl, sgSlc25a1-1 or sgSlc25a1-2 grown in the presence or absence of TGFβ, measured by picrosirius red staining and normalized to the packed cell volume of cells grown on a parallel plate under identical conditions. Values are relative to mock-treated sgCtrl expressing cells.


NIH - 3T3 cells expressing empty vector or Slc25a1 cDNA were treated with TGFβ or mock for 6h , and ROS levels were measured by flow cytometry for CM - H2DCFDA after incubation with CM - H2DCFDA for the last 30 min of the treatment . Values are relative to mock - treated empty vector expressing cells .

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

NIH-3T3 cells expressing empty vector or Slc25a1 cDNA were treated with TGFβ or mock for 6h, and ROS levels were measured by flow cytometry for CM-H2DCFDA after incubation with CM-H2DCFDA for the last 30 min of the treatment. Values are relative to mock-treated empty vector expressing cells.


NIH - 3T3 cells expressing empty vector or Slc25a1 cDNA were treated with TGFβ or mock for 48h , and proline abundance was measured by GC - MS . Values are relative to mock - treated empty vector expressing cells .

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

NIH-3T3 cells expressing empty vector or Slc25a1 cDNA were treated with TGFβ or mock for 48h, and proline abundance was measured by GC-MS. Values are relative to mock-treated empty vector expressing cells.


Western blot of NIH - 3T3 cells expressing empty vector or Slc25a1 cDNA and treated with TGFβ or mock for 48h .

Western blot of NIH-3T3 cells expressing empty vector or Slc25a1 cDNA and treated with TGFβ or mock for 48h.


Collagen abundance in ECM produced by NIH - 3T3 cells expressing empty vector or Slc25a1 cDNA grown in the presence or absence of TGFβ , measured by picrosirius red staining and normalized to the packed cell volume of cells grown on a parallel plate under identical conditions . Values are relative to mock - treated empty vector expressing cells .

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

Collagen abundance in ECM produced by NIH-3T3 cells expressing empty vector or Slc25a1 cDNA grown in the presence or absence of TGFβ, measured by picrosirius red staining and normalized to the packed cell volume of cells grown on a parallel plate under identical conditions. Values are relative to mock-treated empty vector expressing cells.


Analysis of the indicated gene expression datasets for mRNA levels of Slc25a1 , as described in Figure 3 : ( j ) lung tissue from mice with pulmonary fibrosis induced by bleomycin ( Bleo ) treatment compared to saline treatment ( GSE112827 ) ;

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

Analysis of the indicated gene expression datasets for mRNA levels of Slc25a1, as described in Figure 3: (j) lung tissue from mice with pulmonary fibrosis induced by bleomycin (Bleo) treatment compared to saline treatment (GSE112827);


Analysis of the indicated gene expression datasets for mRNA levels of Slc25a1 , as described in Figure 3 : two datasets ( GSE110147 , GSE32537 ) from lungs of patients with idiopathic pulmonary fibrosis ( IPF ) compared to normal controls ( Ctrl ) . AU , arbitrary units . The number of patients per group is indicated .

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

Analysis of the indicated gene expression datasets for mRNA levels of Slc25a1, as described in Figure 3: two datasets (GSE110147, GSE32537) from lungs of patients with idiopathic pulmonary fibrosis (IPF) compared to normal controls (Ctrl). AU, arbitrary units. The number of patients per group is indicated.


Pearson correlation of Slc25a1 and P5CS mRNA levels in IPF patients from GSE32537 . IPF patients from GSE32537 were assigned into P5CSlow / SLC25A1high and P5CShigh / SLC25A1low groups based on the expression level of P5CS and SLC25A1 . " High " represents patients with P5CS or SLC25A1 expression values being above the 75 % percentile of the respective gene expression ; " low " represents patients with expression values being below the 25 % percentile of gene expression . The forced vital capacity ( FVC ) before bronchodilator ( pre - BD ) as percentage of what was predicted for each patient was compared between the groups .

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

Pearson correlation of Slc25a1 and P5CS mRNA levels in IPF patients from GSE32537. IPF patients from GSE32537 were assigned into P5CSlow/SLC25A1high and P5CShigh/SLC25A1low groups based on the expression level of P5CS and SLC25A1. "High" represents patients with P5CS or SLC25A1 expression values being above the 75% percentile of the respective gene expression; "low" represents patients with expression values being below the 25% percentile of gene expression. The forced vital capacity (FVC) before bronchodilator (pre-BD) as percentage of what was predicted for each patient was compared between the groups.


b , c Phospho - sites identified after TiO2 enrichment b and phospho - tyrosine immunoprecipitation ( pY - IP ) c . Left panel : pie chart presenting the proportion of identified phosphorylated serines ( pS ) , threonines ( pT ) and tyrosines ( pY ) in the two data sets . Right panel : cumulative number of unique identified phospho - sites across the biological replicates ( 3 and 4 independent experiments for the TiO2 and pY - IP , respectively ) . Phosphorylated serines , threonines and tyrosines are presented individually next to the total number of identified sites ( pSTY ) . The phospho - sites that were not previously reported in any species in the PhosphoSitePlus database ( www . phosphosite . org ) are reported in isolation in the graph on the right .

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

b, c Phospho-sites identified after TiO2 enrichment b and phospho-tyrosine immunoprecipitation (pY-IP) c. Left panel: pie chart presenting the proportion of identified phosphorylated serines (pS), threonines (pT) and tyrosines (pY) in the two data sets. Right panel: cumulative number of unique identified phospho-sites across the biological replicates (3 and 4 independent experiments for the TiO2 and pY-IP, respectively). Phosphorylated serines, threonines and tyrosines are presented individually next to the total number of identified sites (pSTY). The phospho-sites that were not previously reported in any species in the PhosphoSitePlus database (www.phosphosite.org) are reported in isolation in the graph on the right.


a , b Output of the statistical analyses of the TiO2 - fractionated a and the pY - IP - fractionated b data sets . Left panel : number of phospho - sites significantly regulated upon TCR stimulation presented in bar plots for each time point , next to the total number of regulated sites across the entire time course ( total ) . The number of phosphorylated threonines ( pT ) and serines ( pS ) regulated in the TiO2 data set are indicated in yellow and green , respectively . Right panel : representative volcano plot presenting the statistical significance distribution against the log2 - transformed fold change between 300 seconds and the unstimulated control . For each condition , phospho - sites were considered significantly up - ( red ) or down - ( blue ) regulated when displaying a corrected P - value ≤ 0 . 05 ( Anova test ) and an absolute fold change ≥ 1 . 75 ( see Materials and Methods for more detailed information ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Output of the statistical analyses of the TiO2-fractionated a and the pY-IP-fractionated b data sets. Left panel: number of phospho-sites significantly regulated upon TCR stimulation presented in bar plots for each time point, next to the total number of regulated sites across the entire time course (total). The number of phosphorylated threonines (pT) and serines (pS) regulated in the TiO2 data set are indicated in yellow and green, respectively. Right panel: representative volcano plot presenting the statistical significance distribution against the log2-transformed fold change between 300 seconds and the unstimulated control. For each condition, phospho-sites were considered significantly up- (red) or down- (blue) regulated when displaying a corrected P-value ≤ 0.05 (Anova test) and an absolute fold change ≥ 1.75 (see Materials and Methods for more detailed information).


a Heatmap showing kinase activities based on enrichment of their specific substrates for each cluster . Black dots indicate clusters with significantly enriched kinase activity ( hypergeometric test P - value ≤ 0 . 05 , fold - change ≥ 2 , number of annotated sites ≥ 2 ; Grey squares : fold - change < 1 ) .

a Heatmap showing kinase activities based on enrichment of their specific substrates for each cluster. Black dots indicate clusters with significantly enriched kinase activity (hypergeometric test P-value ≤ 0.05, fold-change ≥ 2, number of annotated sites ≥ 2; Grey squares: fold-change < 1).


d Immunoblot analysis of equal amounts of proteins from total lysates of CD4 + T cells treated with DMSO , U0126 ( 10 μM ) or AKTi VIII ( 10 μM ) and left unstimulated ( - ) or stimulated for 15 , 120 , or 300 sec with anti - CD3 and anti - CD4 antibodies , probed with the indicated phospho - specific antibodies . Anti - ERK1 / 2 immunoblot served as a loading control .

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d Immunoblot analysis of equal amounts of proteins from total lysates of CD4+ T cells treated with DMSO, U0126 (10 μM) or AKTi VIII (10 μM) and left unstimulated (-) or stimulated for 15, 120, or 300 sec with anti-CD3 and anti-CD4 antibodies, probed with the indicated phospho-specific antibodies. Anti-ERK1/2 immunoblot served as a loading control.


e Equal amounts of proteins from total lysates of CD4 + T cells left unstimulated ( - ) or stimulated for 120 , 300 or 600 sec with anti - CD3 and anti - CD4 antibodies were analyzed by immunoblot with antibodies probing the indicated phospho - sites / proteins .

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e Equal amounts of proteins from total lysates of CD4+ T cells left unstimulated (-) or stimulated for 120, 300 or 600 sec with anti-CD3 and anti-CD4 antibodies were analyzed by immunoblot with antibodies probing the indicated phospho-sites/proteins.


b Cas9 - EGFP OT - I CD8 + T cells were transfected with control sgRNA ( sgEGFP ) or with two different sgRNA targeting Itsn2 ( sgITSN2 - 1 or sgITSN2 - 2 ) . Transfected cells were stimulated for 48 h with N4 peptide MHC tetramers ( 0 . 1 nM ) in presence or absence of soluble anti - CD28 antibody or with IL - 7 as control . Cell surface expression of CD69 and CD5 were analyzed by flow cytometry ( Z - score normalization of the Geometric Means of fluorescence MFI per experiment ) in four independent experiments . Comparison between sgEGFP and sgITSN2 conditions were performed using a paired t - test . Boxplot elements : center line corresponds to median , box limits correspond to the first and third quartiles , whiskers indicate variability from Q1 - 1 . 5 . IQR to Q3 + 1 . 5 IQR . Outliers are shown as black dots .

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

b Cas9-EGFP OT-I CD8+ T cells were transfected with control sgRNA (sgEGFP) or with two different sgRNA targeting Itsn2 (sgITSN2-1 or sgITSN2-2). Transfected cells were stimulated for 48 h with N4 peptide MHC tetramers (0.1 nM) in presence or absence of soluble anti-CD28 antibody or with IL-7 as control. Cell surface expression of CD69 and CD5 were analyzed by flow cytometry (Z-score normalization of the Geometric Means of fluorescence MFI per experiment) in four independent experiments. Comparison between sgEGFP and sgITSN2 conditions were performed using a paired t-test. Boxplot elements: center line corresponds to median, box limits correspond to the first and third quartiles, whiskers indicate variability from Q1-1.5. IQR to Q3+1.5 IQR. Outliers are shown as black dots.


b Cas9 - EGFP OT - I CD8 + T cells were transfected with control sgRNA ( sgEGFP ) or with two different sgRNA targeting Itsn2 ( sgITSN2 - 1 or sgITSN2 - 2 ) . Transfected cells were stimulated for 48 h with N4 peptide

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b Cas9-EGFP OT-I CD8+ T cells were transfected with control sgRNA (sgEGFP) or with two different sgRNA targeting Itsn2 (sgITSN2-1 or sgITSN2-2). Transfected cells were stimulated for 48 h with N4 peptide MHC


c Proliferation of OT - I Cas9 - EGFP CD8 + T cells transfected with EGFP or with ITSN2 sgRNA activated for 48 h with N4 peptide MHC tetramers ( 0 . 01 - 0 . 1 nM ) in the presence or absence of soluble anti - CD28 antibody or with PMA and ionomycin ( PMA / Iono ) or with IL - 7 . Data are presented as mean ± standard deviation ( SD ) from four proliferative measures of two independent sgEGFP and sgITSN2 transfections . Data are representative of three independent experiments .

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c Proliferation of OT-I Cas9-EGFP CD8+ T cells transfected with EGFP or with ITSN2 sgRNA activated for 48 h with N4 peptide MHC tetramers (0.01-0.1 nM) in the presence or absence of soluble anti-CD28 antibody or with PMA and ionomycin (PMA/Iono) or with IL-7. Data are presented as mean ± standard deviation (SD) from four proliferative measures of two independent sgEGFP and sgITSN2 transfections. Data are representative of three independent experiments.


d IFN - γ production of cells treated as in c was assessed by intracellular staining after 24 h of stimulation .

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d IFN-γ production of cells treated as in c was assessed by intracellular staining after 24 h of stimulation.


e Proliferation of similar cells as in c activated for 48 h in vitro with a range of increasing doses of N4 peptide MHC tetramers in the presence of soluble anti - CD28 antibody .

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e Proliferation of similar cells as in c activated for 48 h in vitro with a range of increasing doses of N4 peptide MHC tetramers in the presence of soluble anti-CD28 antibody.


a Immunoblot analysis of equal amounts of proteins from total lysates of Cas9 - EGFP OT - I CD8 + T cells transfected with sgEGFP , sgITSN2 - 1 or sgITSN2 - 2 and left unstimulated ( - ) or stimulated for 2 or 5 min with N4 tetramers ( 20 nM ) . Upper panel : membrane probed with antibody to phosphorylated tyrosine ( Anti - p - Tyr ) or anti - LAT ( loading control ) . Lower panel : membrane probed with antibodies to anti - CBL - pY774 , anti - ZAP70 - pY319 / 352 , anti - AKT - pT308 , anti - ERK1 / 2 - pY204 / T202 or anti - ERK1 / 2 ( loading control ) .

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a Immunoblot analysis of equal amounts of proteins from total lysates of Cas9-EGFP OT-I CD8+ T cells transfected with sgEGFP, sgITSN2-1 or sgITSN2-2 and left unstimulated (-) or stimulated for 2 or 5 min with N4 tetramers (20 nM). Upper panel: membrane probed with antibody to phosphorylated tyrosine (Anti-p-Tyr) or anti-LAT (loading control). Lower panel: membrane probed with antibodies to anti-CBL-pY774, anti-ZAP70-pY319/352, anti-AKT-pT308, anti-ERK1/2-pY204/T202 or anti-ERK1/2 (loading control).


b Heat map showing normalized levels ( z - score ) of indicated surface markers and protein phosphorylations ( left margin ) from Cas9 - EGFP OT - I CD8 + T cells transfected with sgEGFP or sgITSN2 - 1 left unstimulated ( - ) or stimulated for 1 , 3 or 10 min with N4 tetramers ( 1 or 10 nM ) and analyzed by mass cytometry . Z - scores were calculated from hyperbolic arcsine ( arcsinh ) transformed intensities .

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b Heat map showing normalized levels (z-score) of indicated surface markers and protein phosphorylations (left margin) from Cas9-EGFP OT-I CD8+ T cells transfected with sgEGFP or sgITSN2-1 left unstimulated (-) or stimulated for 1, 3 or 10 min with N4 tetramers (1 or 10 nM) and analyzed by mass cytometry. Z-scores were calculated from hyperbolic arcsine (arcsinh) transformed intensities.


c Similar cells as in a stimulated with 1 nM of N4 peptide for the indicated time points ( left panel ) or stimulated with increased doses of N4 peptides for 4 h ( right panel ) , were analyzed by FACS for surface TCR expression . Data are presented as mean ± SEM and comparisons were performed using a paired t - test ( * , P ≤ 0 . 05 , * * , P ≤ 0 . 01 ; * * * , P ≤ 0 . 001 ) . A representative analysis of three independent experiments is shown .

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c Similar cells as in a stimulated with 1 nM of N4 peptide for the indicated time points (left panel) or stimulated with increased doses of N4 peptides for 4 h (right panel), were analyzed by FACS for surface TCR expression. Data are presented as mean ± SEM and comparisons were performed using a paired t-test (*, P ≤ 0.05, **, P ≤ 0.01; ***, P ≤ 0.001). A representative analysis of three independent experiments is shown.


d Heatmap showing scaled expression levels ( z - score ) of indicated surface markers and protein phosphorylations ( left margin ) from Cas9 - EGFP OT - I CD8 + T cells transfected with sgEGFP or sgITSN2 - 1 and stimulated for 6 h with increasing doses of N4 peptides or PMA / Ionomycin ( PI ) analyzed by single - cell mass cytometry ( left ) . Z - scores were calculated from hyperbolic arcsine ( arcsinh ) transformed intensities . Signal difference ( mean arcsinh difference ) between sgEGFP and sgITSN2 - 1 transfected cells is depicted on the right panel .

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d Heatmap showing scaled expression levels (z-score) of indicated surface markers and protein phosphorylations (left margin) from Cas9-EGFP OT-I CD8+ T cells transfected with sgEGFP or sgITSN2-1 and stimulated for 6 h with increasing doses of N4 peptides or PMA/Ionomycin (PI) analyzed by single-cell mass cytometry (left). Z-scores were calculated from hyperbolic arcsine (arcsinh) transformed intensities. Signal difference (mean arcsinh difference) between sgEGFP and sgITSN2-1 transfected cells is depicted on the right panel.


Epi - ID H3K79 methylation scores of the deletion mutants of members of the Rpd3L and Rpd3S complexes , calculated as described in Appendix Supplementary Methods , where 0 means a wild - type H3K79me level ( log2 scale ) . The gray dots indicate accessory subunits . UpTag and DownTag are barcode reporters in a promoter and terminator context , respectively . Data was obtained on all Rpd3L / Rpd3S subunits except Sds3 .

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Epi-ID H3K79 methylation scores of the deletion mutants of members of the Rpd3L and Rpd3S complexes, calculated as described in Appendix Supplementary Methods, where 0 means a wild-type H3K79me level (log2 scale). The gray dots indicate accessory subunits. UpTag and DownTag are barcode reporters in a promoter and terminator context, respectively. Data was obtained on all Rpd3L/Rpd3S subunits except Sds3.


Mass spectrometry analysis of H3K79 methylation in wild - type and mutant strains . Mean and individual data points of two biological replicates . p < 0 . 05 ( * ) , p < 0 . 01 ( * * ) and p < 0 . 001 ( * * * ) by two - way ANOVA , comparison to wild type .

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Mass spectrometry analysis of H3K79 methylation in wild-type and mutant strains. Mean and individual data points of two biological replicates. p<0.05 (*), p<0.01 (**) and p<0.001 (***) by two-way ANOVA, comparison to wild type.


Heatmaps of H3K79me1 , H3K79me3 , H2Bub and H3 in wild - type cells , aligned on the TSS . Genes were sorted based on the average H3K79me3 / H3K79me1 ratio in the first 500 bp .

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Heatmaps of H3K79me1, H3K79me3, H2Bub and H3 in wild-type cells, aligned on the TSS. Genes were sorted based on the average H3K79me3/H3K79me1 ratio in the first 500 bp.


Snapshot of depth - normalized ChIP - seq data tracks from wild - type and rpd3Δ strains showing 6 kb surrounding the DBP1 ORF , which is the top gene in the heatmap in panel ( F ) . All tracks have the same y axis ( 0 - 20 RPM ) . A snapshot of another top - regulated gene is shown in Fig EV1D .

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Snapshot of depth-normalized ChIP-seq data tracks from wild-type and rpd3Δ strains showing 6 kb surrounding the DBP1 ORF, which is the top gene in the heatmap in panel (F). All tracks have the same y axis (0-20 RPM). A snapshot of another top-regulated gene is shown in Fig EV1D.


Heatmap of the H3K79me3 / H3 change in rpd3Δ versus wild - type cells , aligned on the TSS . Genes were sorted based on the average ratio in the first 500 bp .

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Heatmap of the H3K79me3/H3 change in rpd3Δ versus wild-type cells, aligned on the TSS. Genes were sorted based on the average ratio in the first 500 bp.


ChIP - seq and RNA - seq data for genes ranked on H3K79me3 / H3 in rpd3Δ / WT , smoothed using locally weighed regression . The gray band around the line shows the 95 % confidence interval . Vertical dashed lines separate 4 groups with distinct changes upon RPD3 deletion . ChIP - seq data of H3K79me1 , H3K79me3 and H2Bub were generated in this study ( plotted is the average coverage in reads per genomic content , RPGC ) , Rpd3 binding , H4ac and WT gene expression data were from ( McKnight et al , 2015 ) , and the relative expression in rpd3Δ / WT from Kemmeren et al ( 2014 ) .

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ChIP-seq and RNA-seq data for genes ranked on H3K79me3/H3 in rpd3Δ/WT, smoothed using locally weighed regression. The gray band around the line shows the 95% confidence interval. Vertical dashed lines separate 4 groups with distinct changes upon RPD3 deletion. ChIP-seq data of H3K79me1, H3K79me3 and H2Bub were generated in this study (plotted is the average coverage in reads per genomic content, RPGC), Rpd3 binding, H4ac and WT gene expression data were from (McKnight et al, 2015), and the relative expression in rpd3Δ/WT from Kemmeren et al (2014).


H3K79me3 / H3 ChIP - qPCR efficiencies ( relative to a non - transcribed region , which was unaffected by RPD3 deletion ) in wild - type and in rpd3Δ cells harboring empty or RPD3 - encoding CEN plasmids . The H188A and H150A - H151A mutations have previously been shown to abrogate catalytic activity ( Kadosh & Struhl , 1998 ) . Error bars indicate standard deviation of three biological replicates . p < 0 . 05 ( * ) , p < 0 . 01 ( * * ) and p < 0 . 001 ( * * * ) by two - way ANOVA , comparison to wild type .

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H3K79me3/H3 ChIP-qPCR efficiencies (relative to a non-transcribed region, which was unaffected by RPD3 deletion) in wild-type and in rpd3Δ cells harboring empty or RPD3-encoding CEN plasmids. The H188A and H150A-H151A mutations have previously been shown to abrogate catalytic activity (Kadosh & Struhl, 1998). Error bars indicate standard deviation of three biological replicates. p<0.05 (*), p<0.01 (**) and p<0.001 (***) by two-way ANOVA, comparison to wild type.


Gene set enrichment analysis on genes ranked on H3K79me3 / H3 in rpd3Δ / WT ; all genes have been ranked and the ranks of the genes in the subsets are indicated by vertical lines . Meiotic ( C ) and Ume6 - bound ( D ) genes are enriched among the genes at which Rpd3 represses H3K79 methylation , and subtelomeric genes ( < 30 kb of telomere ) ( E ) are enriched among genes at which H3K79 methylation is decreased in rpd3Δ cells .

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Gene set enrichment analysis on genes ranked on H3K79me3/H3 in rpd3Δ/WT; all genes have been ranked and the ranks of the genes in the subsets are indicated by vertical lines. Meiotic (C) and Ume6-bound (D) genes are enriched among the genes at which Rpd3 represses H3K79 methylation, and subtelomeric genes (<30 kb of telomere) (E) are enriched among genes at which H3K79 methylation is decreased in rpd3Δ cells.


Mass spectrometry analysis of H3K79 methylation in thymuses from 3 - week - old mice , either wild - type ( Cre - ) or with deleted Hdac1 or Dot1L alleles , as indicated . Mean and individual data points of biological replicates ; H3K79me0 is the predominant state , the y axis is truncated at 70 % for readability . The remaining H3K79 methylation after homozygous Dot1L deletion is probably due to the presence of some cells in which Cre is not expressed ( yet ) . p < 0 . 01 ( * * ) and p < 0 . 001 ( * * * ) by two - way ANOVA , comparison to wild type .

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Mass spectrometry analysis of H3K79 methylation in thymuses from 3-week-old mice, either wild-type (Cre-) or with deleted Hdac1 or Dot1L alleles, as indicated. Mean and individual data points of biological replicates; H3K79me0 is the predominant state, the y axis is truncated at 70% for readability. The remaining H3K79 methylation after homozygous Dot1L deletion is probably due to the presence of some cells in which Cre is not expressed (yet). p<0.01 (**) and p<0.001 (***) by two-way ANOVA, comparison to wild type.


Representative H & E and immunohistochemical stainings on sequential sections of thymic lymphomas of the indicated genotypes . A picture with lower magnification of independent samples is included in Fig EV3B .

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Representative H&E and immunohistochemical stainings on sequential sections of thymic lymphomas of the indicated genotypes. A picture with lower magnification of independent samples is included in Fig EV3B.


Kaplan - Meier curves of mice harboring thymocytes with indicated genotypes . An event was defined as death or sacrifice of a mouse caused by a thymic lymphoma . Mice that died due to other causes or were still alive and event - free at the end of the experiment were censored . Mice for which the cause of death could not be determined were removed from the data . Wild - type mice were the Cre - littermates of the mice that were used for the other curves .

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Kaplan-Meier curves of mice harboring thymocytes with indicated genotypes. An event was defined as death or sacrifice of a mouse caused by a thymic lymphoma. Mice that died due to other causes or were still alive and event-free at the end of the experiment were censored. Mice for which the cause of death could not be determined were removed from the data. Wild-type mice were the Cre- littermates of the mice that were used for the other curves.


Immunoblots showing HDAC1 status and H3K79me / H3K9ac levels in nuclear lysates of Hdac1 - proficient ( p53 - null ) and Hdac1 - deficient thymic lymphoma cell lines . The top four and bottom two panels are from separate lysates of the same cell lines .

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Immunoblots showing HDAC1 status and H3K79me/H3K9ac levels in nuclear lysates of Hdac1-proficient (p53-null) and Hdac1-deficient thymic lymphoma cell lines. The top four and bottom two panels are from separate lysates of the same cell lines.


Mass spectrometry analysis of H3K79 methylation in the cell lines from panel A . Mean and individual data points of two independent cell lines ; H3K79me0 is the predominant state , the y axis is truncated at 80 % for readability . p < 0 . 01 ( * * ) and p < 0 . 001 ( * * * ) by two - way ANOVA .

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Mass spectrometry analysis of H3K79 methylation in the cell lines from panel A. Mean and individual data points of two independent cell lines; H3K79me0 is the predominant state, the y axis is truncated at 80% for readability. p<0.01 (**) and p<0.001 (***) by two-way ANOVA.


Immunoblot showing H2BK120 ubiquitination levels in Hdac1 - proficient and - deficient cell lines ( two independent lines each ) .

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Immunoblot showing H2BK120 ubiquitination levels in Hdac1-proficient and -deficient cell lines (two independent lines each).


Growth curves of Hdac1 - proficient and - deficient cell lines that were left untreated or were infected with empty virus ( pLKO ) or shRNAs against Dot1L and selected with puromycin . Growth curves were determined by a series of resazurin assays , which measure metabolic activity , starting four days post - infection . Error bars indicate the range of two replicates from independent cell lines .

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Growth curves of Hdac1-proficient and -deficient cell lines that were left untreated or were infected with empty virus (pLKO) or shRNAs against Dot1L and selected with puromycin. Growth curves were determined by a series of resazurin assays, which measure metabolic activity, starting four days post-infection. Error bars indicate the range of two replicates from independent cell lines.


Inhibitor dose - response curves of the two DOT1L inhibitors EPZ - 5676 ( Pinometostat ) and SGC - 0946 in Hdac1 - proficient and - deficient cell lines . Cell viability was measured by a resazurin assay after three days of treatment and measurements were normalized to DMSO - treated cells . Two independent cell lines are plotted separately ; error bars indicate the range of two biological replicates .

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

Inhibitor dose-response curves of the two DOT1L inhibitors EPZ-5676 (Pinometostat) and SGC-0946 in Hdac1-proficient and -deficient cell lines. Cell viability was measured by a resazurin assay after three days of treatment and measurements were normalized to DMSO-treated cells. Two independent cell lines are plotted separately; error bars indicate the range of two biological replicates.


Representative apoptosis FACS plots of cell lines treated with DMSO or the DOT1L inhibitor SGC - 0946 for two days . Annexin V staining and DAPI staining were performed on unpermeabilized cells to distinguish live ( Annexin V low ; DAPI low ) , apoptotic ( Annexin V high ; DAPI low ) and dead ( Annexin V high ; DAPI high ) cells . Quantification of several independent FACS experiments , including the experiment shown in panel ( A ) . Mean with individual data points of 2 - 4 replicates each of two independent lines per genotype . p < 0 . 001 ( * * * ) by two - way ANOVA , comparison to corresponding DMSO control .

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Representative apoptosis FACS plots of cell lines treated with DMSO or the DOT1L inhibitor SGC-0946 for two days. Annexin V staining and DAPI staining were performed on unpermeabilized cells to distinguish live (Annexin V low; DAPI low), apoptotic (Annexin V high; DAPI low) and dead (Annexin V high; DAPI high) cells. Quantification of several independent FACS experiments, including the experiment shown in panel (A). Mean with individual data points of 2-4 replicates each of two independent lines per genotype. p<0.001 (***) by two-way ANOVA, comparison to corresponding DMSO control.


( B ) The inherent elastic response of a lipid membrane ( POPC ) was studied by enforcing indentations via the isolated SNARE C - termini in the presence or absence of a short linker peptide , or the linker plus a fluorescent protein tag ( FP ) . We define " relative indentation " as the change in C - terminus ( Nyv1 ) - C - terminus ( Vam3 ) distance that occurs when the SNARE complex performs mechanical work . It reflects the depth of the formed well upon squeezing . The slope of the obtained work profile estimates the required force ( thin black line ) . Perforation ( red arrow ) requires a force of 80 pN for the wild - type C - termini . The plateau within the work profile indicates that the membrane gives up its elastic response ( perforation transition ) . C - terminal tags oppose such a perforation transition but do not significantly affect the forces observed within the indentation regime . Furthermore , if the unstructured peptide - spacer would be alpha - helical - and thus much shorter - indentation forces would still be only slightly affected .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) The inherent elastic response of a lipid membrane (POPC) was studied by enforcing indentations via the isolated SNARE C-termini in the presence or absence of a short linker peptide, or the linker plus a fluorescent protein tag (FP). We define "relative indentation" as the change in C-terminus (Nyv1) - C-terminus (Vam3) distance that occurs when the SNARE complex performs mechanical work. It reflects the depth of the formed well upon squeezing. The slope of the obtained work profile estimates the required force (thin black line). Perforation (red arrow) requires a force of 80 pN for the wild-type C-termini. The plateau within the work profile indicates that the membrane gives up its elastic response (perforation transition). C-terminal tags oppose such a perforation transition but do not significantly affect the forces observed within the indentation regime. Furthermore, if the unstructured peptide-spacer would be alpha-helical - and thus much shorter - indentation forces would still be only slightly affected.


( B ) Vacuoles were isolated from the indicated yeast strains and 30 µg of proteins analyzed by SDS - PAGE and Western blotting .

O 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) Vacuoles were isolated from the indicated yeast strains and 30 µg of proteins analyzed by SDS-PAGE and Western blotting.


( C ) In vivo vacuole morphology was assayed by FM4 - 64 staining . Scale bar : 5 µm . ( D ) The cells were grouped into three categories according to the number of vacuoles visible per 100 cells . Values represent the means and s . d . from three independent experiments .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) In vivo vacuole morphology was assayed by FM4-64 staining. Scale bar: 5 µm. (D) The cells were grouped into three categories according to the number of vacuoles visible per 100 cells. Values represent the means and s. d. from three independent experiments.


( E ) Vacuoles were isolated from BJ3505 strains expressing the indicated tagged versions of Vam3 and Nyv1 and Pho8 - EGFP or Pho8 - mCherry . 10 µg of vacuoles were incubated in standard fusion reactions in the presence or absence of ATP and analyzed by confocal microscopy . Arrows indicate examples of fusion products . Scale bar : 5 µm . ( F ) Fusion activity : vacuole fusion was assayed by measuring the percentage of co - localization of the two Pho8 - EGFP and Pho8 - mCherry signals . Means s . d . are shown for at least 100 stained vacuoles from 3 experiments .

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

(E) Vacuoles were isolated from BJ3505 strains expressing the indicated tagged versions of Vam3 and Nyv1 and Pho8-EGFP or Pho8-mCherry. 10 µg of vacuoles were incubated in standard fusion reactions in the presence or absence of ATP and analyzed by confocal microscopy. Arrows indicate examples of fusion products. Scale bar: 5 µm. (F) Fusion activity: vacuole fusion was assayed by measuring the percentage of co-localization of the two Pho8-EGFP and Pho8-mCherry signals. Means s.d. are shown for at least 100 stained vacuoles from 3 experiments.


( A ) Vacuoles were isolated from BJ3505 cells expressing the indicated versions of Vam3 and / or Nyv1 . 10 µg of the organelles were analyzed by SDS - PAGE and Western blotting using antibodies to Vti1 , Vam3 and Nyv1 .

O O O O O O O O O O O O O O O O O O O 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) Vacuoles were isolated from BJ3505 cells expressing the indicated versions of Vam3 and/or Nyv1. 10 µg of the organelles were analyzed by SDS-PAGE and Western blotting using antibodies to Vti1, Vam3 and Nyv1.


( B ) Localization in vivo . The indicated strains were grown logarithmically in synthetic complete ( SC ) medium . After 16 h at 30 ° C , when the final OD of each culture was about 1 , the cells were observed by confocal fluorescence and differential interference ( DIC ) microscopy . Scale bar : 5 µm .

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

(B) Localization in vivo. The indicated strains were grown logarithmically in synthetic complete (SC) medium. After 16 h at 30°C, when the final OD of each culture was about 1, the cells were observed by confocal fluorescence and differential interference (DIC) microscopy. Scale bar: 5 µm.


( A ) In vivo morphology . Cells expressing the indicated SNARE variants were grown in SC medium ( 16 h , 30 ° C ) , stained with FM4 - 64 and their relative morphology was assessed by fluorescence and DIC microscopy . Scale bar : 5 µm . The cells were grouped into three categories according to the number of vacuoles visible per 100 cells . Values represent the means and s . d . from three independent experiments .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) In vivo morphology. Cells expressing the indicated SNARE variants were grown in SC medium (16 h, 30°C), stained with FM4-64 and their relative morphology was assessed by fluorescence and DIC microscopy. Scale bar: 5 µm. The cells were grouped into three categories according to the number of vacuoles visible per 100 cells. Values represent the means and s. d. from three independent experiments.


( B ) and ( C ) SNARE activation on isolated vacuoles . Vacuoles were isolated from a strain co - expressing NYV1 - S9 - EGFP and VAM3 - S9 - mCitrine and from an isogenic wild - type . 150 µg of the organelles were incubated in fusion reactions in the presence or absence of an ATP - regenerating system and recombinant , purified Sec18 / NSF ( rSec18 , 50 µg / ml ) . After 10 min of incubation at 27 ° C , the vacuoles were solubilized and immunoprecipitated with antibodies to Nyv1 . Co - immunoprecipitated proteins analyzed by SDS - PAGE and Western blotting . The histograms provide quantifications of the band intensities as the means and s . d . from three independent experiments .

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

(B) and (C) SNARE activation on isolated vacuoles. Vacuoles were isolated from a strain co-expressing NYV1-S9-EGFP and VAM3-S9-mCitrine and from an isogenic wild-type. 150 µg of the organelles were incubated in fusion reactions in the presence or absence of an ATP-regenerating system and recombinant, purified Sec18/NSF (rSec18, 50 µg/ml). After 10 min of incubation at 27°C, the vacuoles were solubilized and immunoprecipitated with antibodies to Nyv1. Co-immunoprecipitated proteins analyzed by SDS-PAGE and Western blotting. The histograms provide quantifications of the band intensities as the means and s.d. from three independent experiments.


( A ) Vacuoles were isolated from the indicated yeast strains and 30 µg of proteins analyzed by SDS - PAGE and Western blotting .

(A) Vacuoles were isolated from the indicated yeast strains and 30 µg of proteins analyzed by SDS-PAGE and Western blotting.


( B ) Vacuole morphology was assessed for the indicated cells as in Fig . 3C . Scale bar : 5 µm .

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

(B) Vacuole morphology was assessed for the indicated cells as in Fig. 3C. Scale bar: 5 µm.


( C ) Vacuoles were isolated from BJ3505 and DKY6281 cells with SNAREs tagged or deleted as indicated . Note that presence of Nyv1 on only one fusion partner still permits efficient fusion [ 65 ] but ensures that trans - SNARE complexes can only form in one orientation , between the Q - SNARE in DKY6281 and the R - SNARE in BJ3505 . The vacuoles were used in standard fusion reactions and content mixing was measured via the alkaline phosphatase assay . In parallel , identical samples were incubated either incubated on ice , which prevents fusion , or in the presence of 0 . 5 % Triton X - 100 , which allows fusion - independent access of the maturase Pep4 to pro - ALP and controls for the levels of these two reporter enzymes in the samples .

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

(C) Vacuoles were isolated from BJ3505 and DKY6281 cells with SNAREs tagged or deleted as indicated. Note that presence of Nyv1 on only one fusion partner still permits efficient fusion [65] but ensures that trans-SNARE complexes can only form in one orientation, between the Q-SNARE in DKY6281 and the R-SNARE in BJ3505. The vacuoles were used in standard fusion reactions and content mixing was measured via the alkaline phosphatase assay. In parallel, identical samples were incubated either incubated on ice, which prevents fusion, or in the presence of 0.5% Triton X-100, which allows fusion-independent access of the maturase Pep4 to pro-ALP and controls for the levels of these two reporter enzymes in the samples.


( D ) Using strains from C , lipid and content mixing ( left and right panel , respectively ) were performed in parallel in the presence or absence of ATP . Anti - Vam3 antibody ( 3 µg ) was added to some reactions in order to inhibit trans - SNARE pairing and fusion . Means and the s . d . are shown for three independent experiments .

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

(D) Using strains from C, lipid and content mixing (left and right panel, respectively) were performed in parallel in the presence or absence of ATP. Anti-Vam3 antibody (3 µg) was added to some reactions in order to inhibit trans-SNARE pairing and fusion. Means and the s.d. are shown for three independent experiments.


Vam3 and Nyv1 were tagged with mCitrine and EGFP as shown Fig . 3A but the spacer was extended ( S34 ) by an additional 25 - amino acid sequence ( SGGGGSGGGGSGGGGSGGGGAAAGG ) to the previous short spacer . ( A ) Protein levels on isolated vacuoles from these strains were compared as in Fig . 3B .

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

Vam3 and Nyv1 were tagged with mCitrine and EGFP as shown Fig. 3A but the spacer was extended (S34) by an additional 25-amino acid sequence (SGGGGSGGGGSGGGGSGGGGAAAGG) to the previous short spacer. (A) Protein levels on isolated vacuoles from these strains were compared as in Fig. 3B.


Vam3 and Nyv1 were tagged with mCitrine and EGFP as shown Fig . 3A but the spacer was extended ( S34 ) by an additional 25 - amino acid sequence ( SGGGGSGGGGSGGGGSGGGGAAAGG ) to the previous short spacer . ( B ) Vacuole morphology was assessed as in Fig . 3C . Scale bar : 5 µm . ( C ) The cells were grouped into three categories according to the number of vacuoles visible per 100 cells . Values represent the means and s . d . from three independent experiments .

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

Vam3 and Nyv1 were tagged with mCitrine and EGFP as shown Fig. 3A but the spacer was extended (S34) by an additional 25-amino acid sequence (SGGGGSGGGGSGGGGSGGGGAAAGG) to the previous short spacer. (B) Vacuole morphology was assessed as in Fig. 3C. Scale bar: 5 µm. (C) The cells were grouped into three categories according to the number of vacuoles visible per 100 cells. Values represent the means and s. d. from three independent experiments.


Vam3 and Nyv1 were tagged with mCitrine and EGFP as shown Fig . 3A but the spacer was extended ( S34 ) by an additional 25 - amino acid sequence ( SGGGGSGGGGSGGGGSGGGGAAAGG ) to the previous short spacer . ( D ) Fusion activity : vacuoles were isolated from BJ3505 strains expressing the indicated versions of Vam3 and Nyv1 and Pho8 - mCFP or Pho8 - mCherry . 10 µg of vacuoles were incubated in standard fusion reactions in the presence or absence of ATP and analyzed by confocal microscopy . Arrows indicate examples of fusion products . Means s . d . are shown for at least 100 stained vacuoles from 3 experiments . Scale bar : 5 µm . ( E ) Vacuole fusion was assayed by measuring the percentage of co - localisation of the two Pho8 - mCFP and Pho8 - mCherry signals .

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

Vam3 and Nyv1 were tagged with mCitrine and EGFP as shown Fig. 3A but the spacer was extended (S34) by an additional 25-amino acid sequence (SGGGGSGGGGSGGGGSGGGGAAAGG) to the previous short spacer. (D) Fusion activity: vacuoles were isolated from BJ3505 strains expressing the indicated versions of Vam3 and Nyv1 and Pho8-mCFP or Pho8-mCherry. 10 µg of vacuoles were incubated in standard fusion reactions in the presence or absence of ATP and analyzed by confocal microscopy. Arrows indicate examples of fusion products. Means s.d. are shown for at least 100 stained vacuoles from 3 experiments. Scale bar: 5 µm. (E) Vacuole fusion was assayed by measuring the percentage of co-localisation of the two Pho8-mCFP and Pho8-mCherry signals.


( A ) Yeast strain expressing a plasmid with Vph1 - GFP was grown in SC - URA medium for 16 h at 30 ° C . The mobility of Vph1 - GFP in the absence ( upper panels ) or presence ( lower panel ) of CPZ ( 150 µM ) was assessed by FRAP . ( B ) The histogram shows the GFP signals in the bleached area during the FRAP procedure . ( C ) The half - time is shown . Means and s . d . are shown for 20 cells of 3 independent experiments .

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

(A) Yeast strain expressing a plasmid with Vph1-GFP was grown in SC-URA medium for 16 h at 30°C. The mobility of Vph1-GFP in the absence (upper panels) or presence (lower panel) of CPZ (150 µM) was assessed by FRAP. (B) The histogram shows the GFP signals in the bleached area during the FRAP procedure. (C) The half-time is shown. Means and s.d. are shown for 20 cells of 3 independent experiments.


( D ) Yeast cells were grown in SC medium for 16 h at 30 ° C . After staining with FM4 - 64 , cells were treated with CPZ ( 150 µM ) . Vacuole morphology was assessed by confocal microscopy before and after 5 and 20 min of CPZ addition . Scale bar : 5 µm .

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

(D) Yeast cells were grown in SC medium for 16 h at 30°C. After staining with FM4-64, cells were treated with CPZ (150 µM). Vacuole morphology was assessed by confocal microscopy before and after 5 and 20 min of CPZ addition. Scale bar: 5 µm.


( E ) Fusion activity . Vacuoles were isolated from BJ cells carrying Nyv1 - S9 - EGFP and DKY cells deleted for Nyv1 and carrying Vam3 - S9 - mCitrine or from wild - type cells and incubated in standard fusion reactions in the presence or absence of chlorpromazine ( CPZ , 150 µM ) and anti - Vam3 ( 3 µg ) . Content mixing was assayed via alkaline phosphatase activity . Means and s . d . are shown for three independent experiments .

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

(E) Fusion activity. Vacuoles were isolated from BJ cells carrying Nyv1-S9-EGFP and DKY cells deleted for Nyv1 and carrying Vam3-S9-mCitrine or from wild-type cells and incubated in standard fusion reactions in the presence or absence of chlorpromazine (CPZ, 150 µM) and anti-Vam3 (3 µg). Content mixing was assayed via alkaline phosphatase activity. Means and s.d. are shown for three independent experiments.


Electropherograms of the XRCC4 genomic region encompassing the nucleotide substitutions in available members of the family .

Electropherograms of the XRCC4 genomic region encompassing the nucleotide substitutions in available members of the family.


Quantitative real - time PCR of XRCC4 relative to GAPDH mRNA in affected ( II - 1 and II - 2 ) and control ( CT1 and CT2 ) fibroblasts . Each value refers to the mean of three independent experiments performed in duplicate . Error bars indicate the standard deviation .

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

Quantitative real-time PCR of XRCC4 relative to GAPDH mRNA in affected (II-1 and II-2) and control (CT1 and CT2) fibroblasts. Each value refers to the mean of three independent experiments performed in duplicate. Error bars indicate the standard deviation.


Immunoblot analysis of total lysates from fibroblasts ( fbs ) and of in vitro - synthesized proteins ( i . v . p ) . fl : in vitro - translated full - length XRCC4 protein ; ret . : reticulocyte lysate used for in vitro protein synthesis ; 225 * : in vitro - translated XRCC4 R225 * truncated protein ; Ct : lysate of control fbs ; and II - 1 and II - 2 : lysates of fbs from patient II - 1 and II - 2 . α - XRCC4 ( Abcam , 1 : 1 , 000 ) and α - GAPDH antibodies were used .

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

Immunoblot analysis of total lysates from fibroblasts (fbs) and of in vitro-synthesized proteins (i.v.p). fl: in vitro-translated full-length XRCC4 protein; ret.: reticulocyte lysate used for in vitro protein synthesis; 225*: in vitro-translated XRCC4 R225* truncated protein; Ct: lysate of control fbs; and II-1 and II-2: lysates of fbs from patient II-1 and II-2. α-XRCC4 (Abcam, 1:1,000) and α-GAPDH antibodies were used.


Immunoblot analysis of total lysates from fibroblasts using α - LIG4 ( GeneTex , 1 : 1 , 000 ) and α - SDHA antibodies . Ct : lysate of control fbs ; II - 1 and II - 2 : lysates of fbs from patient II - 1 and II - 2 . The position of the molecular weight ( MW ) marker proteins is indicated .

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

Immunoblot analysis of total lysates from fibroblasts using α-LIG4 (GeneTex, 1:1,000) and α-SDHA antibodies. Ct: lysate of control fbs; II-1 and II-2: lysates of fbs from patient II-1 and II-2. The position of the molecular weight (MW) marker proteins is indicated.


γ - H2AX nuclear foci in XRCC4wt and mutant fibroblasts ( XRCC4m / m1 and XRCC4m / m2 ) were determined at different times after irradiation with 0 . 5 Gy of γ - rays . Values , subtracted of their non - irradiated control ( 1 . 3 foci / nucleus in XRCC4wt cells and 1 . 4 and 0 . 9 foci / nucleus in XRCC4m / m1 and XRCC4m / m2 cells , respectively ) , are the mean ± SD of three independent experiments . Percentages of γ - H2AX foci in XRCC4wt and XRCC4m / m fibroblasts ( pooled values ) remaining at the indicated time - points . Immunofluorescence micrographs of γ - H2AX foci in XRCC4wt and XRCC4m / m fibroblasts . Data information : * * * P < 0 . 001 ; * * P < 0 . 01 ; * P < 0 . 05 ( XRCC4m / m vs . XRCC4wt cells ) , two - way ANOVA , Bonferroni post hoc 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

γ-H2AX nuclear foci in XRCC4wt and mutant fibroblasts (XRCC4m/m1 and XRCC4m/m2) were determined at different times after irradiation with 0.5 Gy of γ-rays. Values, subtracted of their non-irradiated control (1.3 foci/nucleus in XRCC4wt cells and 1.4 and 0.9 foci/nucleus in XRCC4m/m1 and XRCC4m/m2 cells, respectively), are the mean ± SD of three independent experiments.Percentages of γ-H2AX foci in XRCC4wt and XRCC4m/m fibroblasts (pooled values) remaining at the indicated time-points.Immunofluorescence micrographs of γ-H2AX foci in XRCC4wt and XRCC4m/m fibroblasts.Data information: ***P < 0.001; **P < 0.01; *P < 0.05 (XRCC4m/m vs. XRCC4wt cells), two-way ANOVA, Bonferroni post hoc test.


A , B Percentages of γ - H2AX nuclear foci remaining in CENP - F - negative ( A ) and CENP - F - positive ( B ) cells at the indicated time - points after irradiation with 0 . 5 Gy of γ - rays . Values , subtracted of their non - irradiated controls , are the mean ± SD of three independent experiments . * * * P < 0 . 001 ; * P < 0 . 05 ( XRCC4m / m versus XRCC4wt cells ) , two - way ANOVA , Bonferroni post hoc test . C Immunofluorescence micrographs of γ - H2AX foci in CENP - F - positive and negative fibroblasts at 2 h after irradiation . Arrow heads indicate the CENP - F - positive cells .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Percentages of γ-H2AX nuclear foci remaining in CENP-F-negative (A) and CENP-F-positive (B) cells at the indicated time-points after irradiation with 0.5 Gy of γ-rays. Values, subtracted of their non-irradiated controls, are the mean ± SD of three independent experiments. ***P < 0.001; *P < 0.05 (XRCC4m/m versus XRCC4wt cells), two-way ANOVA, Bonferroni post hoc test.C Immunofluorescence micrographs of γ-H2AX foci in CENP-F-positive and negative fibroblasts at 2 h after irradiation. Arrow heads indicate the CENP-F-positive cells.


RAD51 foci in XRCC4wt and XRCC4m / m fibroblasts irradiated with 0 . 5 Gy of γ - rays . Values are the mean ± SD of three independent experiments . * * * P < 0 . 001 ; * * P < 0 . 01 ( XRCC4m / m versus XRCC4wt cells ) , two - way ANOVA , Bonferroni post hoc test . Immunofluorescence micrographs of RAD51 foci in XRCC4wt and XRCC4m / mfibroblasts . Arrowheads indicate the CENP - F - positive cells .

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

RAD51 foci in XRCC4wt and XRCC4m/m fibroblasts irradiated with 0.5 Gy of γ-rays. Values are the mean ± SD of three independent experiments. ***P < 0.001; **P < 0.01 (XRCC4m/m versus XRCC4wt cells), two-way ANOVA, Bonferroni post hoc test.Immunofluorescence micrographs of RAD51 foci in XRCC4wt and XRCC4m/mfibroblasts. Arrowheads indicate the CENP-F-positive cells.


XRCC4wt and XRCC4m / m cells irradiated with 0 . 5 Gy of γ - rays were exposed to the PARP1 inhibitor 3 ′ - AB or to solvent ( DMSO ) . The γ - H2AX foci number is the mean ± SD of three independent experiments . * P < 0 . 05 ( XRCC4m / m + 3 ′ - AB versus XRCC4m / m + DMSO ) , * * * P < 0 . 001 ( XRCC4m / m + 3 ′ - AB versus XRCC4wt + 3 ′ - AB ) , two - way ANOVA , Bonferroni post hoc test .

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

XRCC4wt and XRCC4m/m cells irradiated with 0.5 Gy of γ-rays were exposed to the PARP1 inhibitor 3′-AB or to solvent (DMSO). The γ-H2AX foci number is the mean ± SD of three independent experiments. *P < 0.05 (XRCC4m/m + 3′-AB versus XRCC4m/m + DMSO), ***P < 0.001 (XRCC4m/m + 3′-AB versus XRCC4wt + 3′-AB), two-way ANOVA, Bonferroni post hoc test.


A , B Percentages of DSBs repaired in increasing time intervals ( 0 . 5 - 2 h , 0 . 5 - 6 h , 0 . 5 - 24 h ) were calculated in XRCC4wt ( A ) and XRCC4m / m ( B ) cells from the number of γ - H2AX foci in presence or absence of a PARP - 1 inhibitor , 3 ′ - AB . * * P < 0 . 01 ( XRCC4m / m + 3 ′ - AB versus XRCC4m / m + DMSO ) , two - way ANOVA , Bonferroni post hoc 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O

A, B Percentages of DSBs repaired in increasing time intervals (0.5-2 h, 0.5-6 h, 0.5-24 h) were calculated in XRCC4wt (A) and XRCC4m/m (B) cells from the number of γ-H2AX foci in presence or absence of a PARP-1 inhibitor, 3′-AB. **P < 0.01 (XRCC4m/m + 3′-AB versus XRCC4m/m + DMSO), two-way ANOVA, Bonferroni post hoc test.


Vero and Caco - 2 cells were infected with SARS - CoV - 2 at MOIs of 0 . 2 for 8 h . Infected cells were then permeabilized and immunostained for the intracellular SARS - CoV - 2 nucleoprotein ( NP , red ) . Nuclei were stained with Hoechst ( blue ) before imaging by fluorescence confocal microscopy . Scale bars : 100 µm .

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Vero and Caco-2 cells were infected with SARS-CoV-2 at MOIs of 0.2 for 8 h. Infected cells were then permeabilized and immunostained for the intracellular SARS-CoV-2 nucleoprotein (NP, red). Nuclei were stained with Hoechst (blue) before imaging by fluorescence confocal microscopy. Scale bars: 100 µm.


Infection of Vero and Caco - 2 cells was monitored over 24 h using the same flow cytometry - based assay used for the experiment shown in panel D . Infection is given as the total fluorescence associated with the NP protein - positive cells . MFI , mean fluorescence intensity . n = 3 .

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Infection of Vero and Caco-2 cells was monitored over 24 h using the same flow cytometry-based assay used for the experiment shown in panel D. Infection is given as the total fluorescence associated with the NP protein-positive cells. MFI, mean fluorescence intensity. n = 3.


Cells were pretreated with the indicated concentrations of aprotinin ( A ) and SB412515 ( B ) , which are inhibitors of TMPRSS2 and cathepsin L , respectively . Infection of Calu - 3 , Caco - 2 , A549 * , and Vero cells with SARS - CoV - 2 at MOIs of 0 . 3 , 0 . 4 , 0 . 2 , and 0 . 3 , respectively , was achieved in the continuous presence of the drug . Infected cells were quantified by flow cytometry as described in Fig . 1D , and data were normalized to samples where inhibitors had been omitted . n = 2 - 4 biological replicates .

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Cells were pretreated with the indicated concentrations of aprotinin (A) and SB412515 (B), which are inhibitors of TMPRSS2 and cathepsin L, respectively. Infection of Calu-3, Caco-2, A549*, and Vero cells with SARS-CoV-2 at MOIs of 0.3, 0.4, 0.2, and 0.3, respectively, was achieved in the continuous presence of the drug. Infected cells were quantified by flow cytometry as described in Fig. 1D, and data were normalized to samples where inhibitors had been omitted. n = 2-4 biological replicates.


SARS - CoV - 2 particles were bound to A549 * and Vero cells ( MOIs 0 . 2 and 0 . 3 , respectively ) ( C ) or Calu - 3 and Caco - 2 cells ( 0 . 6 and 0 . 5 , respectively ) ( D ) on ice for 90 min and subsequently warmed rapidly to 37 ° C to allow infectious penetration . SB412515 ( 10 µM , C ) or aprotinin ( 30 µM , D ) was added at different times post warming to block further proteolytic activation . Infection was analyzed by flow cytometry , and data were normalized to samples where protease inhibitors had been omitted . n = 2 .

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SARS-CoV-2 particles were bound to A549* and Vero cells (MOIs 0.2 and 0.3, respectively) (C) or Calu-3 and Caco-2 cells (0.6 and 0.5, respectively) (D) on ice for 90 min and subsequently warmed rapidly to 37°C to allow infectious penetration. SB412515 (10 µM, C) or aprotinin (30 µM, D) was added at different times post warming to block further proteolytic activation. Infection was analyzed by flow cytometry, and data were normalized to samples where protease inhibitors had been omitted. n = 2.


Cells were pretreated with endosomal - pH interfering drugs at the indicated concentrations and subsequently infected with SARS - CoV - 2 as described in Fig . 2A and 2B in the continuous presence of NH4Cl ( A ) , chloroquine ( B ) , bafilomycin A1 ( C ) , or concanamycin B ( D ) . The proportion of infected cells was quantified by flow cytometry as described in Fig . 1D , and data were normalized to control samples that were not treated with inhibitors . n = 2 - 6 biological replicates .

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Cells were pretreated with endosomal-pH interfering drugs at the indicated concentrations and subsequently infected with SARS-CoV-2 as described in Fig. 2A and 2B in the continuous presence of NH4Cl (A), chloroquine (B), bafilomycin A1 (C), or concanamycin B (D). The proportion of infected cells was quantified by flow cytometry as described in Fig. 1D, and data were normalized to control samples that were not treated with inhibitors. n = 2-6 biological replicates.


A549 * cells expressing or lacking TMPRSS2 were pretreated with the indicated concentrations of camostat mesylate ( B ) , SB412515 ( C ) , and bafilomycin A1 ( D ) . Infection with SARS - CoV - 2 ( MOI ~ 0 . 2 ) was achieved in the continuous presence of the drug . Infected cells were quantified by flow cytometry as described in Fig . 1D , and data were normalized to samples where inhibitors had been omitted . n = 5 - 6 biological replicates .

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A549* cells expressing or lacking TMPRSS2 were pretreated with the indicated concentrations of camostat mesylate (B), SB412515 (C), and bafilomycin A1 (D). Infection with SARS-CoV-2 (MOI ~0.2) was achieved in the continuous presence of the drug. Infected cells were quantified by flow cytometry as described in Fig. 1D, and data were normalized to samples where inhibitors had been omitted. n = 5-6 biological replicates.


The timing of the MG - 132 - sensitive step during SARS - CoV - 2 infectious entry into Calu - 3 and Caco - 2 cells was assayed as detailed in D but using 60 µM MG - 132 for Caco - 2 cells and MOIs of 0 . 5 and 0 . 6 , respectively . n = 2 .

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The timing of the MG-132-sensitive step during SARS-CoV-2 infectious entry into Calu-3 and Caco-2 cells was assayed as detailed in D but using 60 µM MG-132 for Caco-2 cells and MOIs of 0.5 and 0.6, respectively. n = 2.


A549 * cells expressing or lacking TMPRSS2 were pretreated with MG - 132 at the indicated concentrations and subsequently infected with SARS - CoV - 2 ( MOI ~ 0 . 2 ) in the continuous presence of the drug . The proportion of infected cells was quantified by flow cytometry as described in Fig . 1D , and data were normalized to that from control samples for which MG - 132 had been omitted . n = 6 biological replicates .

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A549* cells expressing or lacking TMPRSS2 were pretreated with MG-132 at the indicated concentrations and subsequently infected with SARS-CoV-2 (MOI ~0.2) in the continuous presence of the drug. The proportion of infected cells was quantified by flow cytometry as described in Fig. 1D, and data were normalized to that from control samples for which MG-132 had been omitted. n = 6 biological replicates.


SARS - CoV - 2 was subjected to pretreatment with trypsin and furin for 15 min at 37 ° C prior to infection of Caco - 2 and Vero cells ( MOIs of 0 . 4 and 0 . 3 , respectively ) . Infected cells were quantified by flow cytometry as described in Fig . 1D . Data were normalized to samples not pretreated with trypsin . n = 4 - 6 biological replicates .

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SARS-CoV-2 was subjected to pretreatment with trypsin and furin for 15 min at 37°C prior to infection of Caco-2 and Vero cells (MOIs of 0.4 and 0.3, respectively). Infected cells were quantified by flow cytometry as described in Fig. 1D. Data were normalized to samples not pretreated with trypsin. n = 4-6 biological replicates.


A549 * expressing or lacking TMPRSS2 cells and Vero cells were first infected with SARS - CoV - 2 at MOIs of 0 . 1 and 0 . 2 , respectively , for 24 h and then cocultured for 5 h along with target cells , not infected but prestained with CMFDA , a cytosolic green dye . Cells were subsequently treated with trypsin or furin for 5 min at 37 ° C and left to coculture for an additional hour at 37 ° C . After fixation , nuclei were stained with Hoechst ( blue ) , and infected cells were subjected to immunofluorescence staining against SARS - CoV - 2 nucleoprotein ( magenta ) . Samples were ultimately imaged by confocal fluorescence microscopy . White stars indicate syncytia with at least four nuclei . Scale bars : 100 µm . Images of microscope fields obtained in ( E ) were quantified [ A549 * : n ( no virus ) = 30 , n ( no protease ) = 80 , n ( trypsin ) = 80 , and n ( furin ) = 60 ; A549 * TMPRSS2 : n ( no virus ) = 30 , n ( no protease ) = 80 , n ( trypsin ) = 79 , and n ( furin ) = 60 ; Vero : n ( no virus ) = 40 , n ( no protease ) = 112 , n ( trypsin ) = 114 , and n ( furin ) = 60 ] . The fusion index is given as f = 1 - [ ( number of cells in a field after fusion ) / ( number of nuclei ) ] .

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

A549* expressing or lacking TMPRSS2 cells and Vero cells were first infected with SARS-CoV-2 at MOIs of 0.1 and 0.2, respectively, for 24 h and then cocultured for 5 h along with target cells, not infected but prestained with CMFDA, a cytosolic green dye. Cells were subsequently treated with trypsin or furin for 5 min at 37°C and left to coculture for an additional hour at 37°C. After fixation, nuclei were stained with Hoechst (blue), and infected cells were subjected to immunofluorescence staining against SARS-CoV-2 nucleoprotein (magenta). Samples were ultimately imaged by confocal fluorescence microscopy. White stars indicate syncytia with at least four nuclei. Scale bars: 100 µm. Images of microscope fields obtained in (E) were quantified [A549*: n (no virus) = 30, n (no protease) = 80, n (trypsin) = 80, and n (furin) = 60; A549* TMPRSS2: n (no virus) = 30, n (no protease) = 80, n (trypsin) = 79, and n (furin) = 60; Vero: n (no virus) = 40, n (no protease) = 112, n (trypsin) = 114, and n (furin) = 60]. The fusion index is given as f = 1 - [(number of cells in a field after fusion)/(number of nuclei)].


SARS - CoV - 2 particles ( MOI of 0 . 3 ) were first subjected to trypsin treatment for 15 min at 37 ° C and then exposed to buffers at the indicated pH for 10 min at 37 ° C , and vice versa . Calu - 3 and Vero cells were then infected at a MOI of 0 . 3 and analyzed by flow cytometry as described in Fig . 1D . n = 4 - 8 biological replicates .

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SARS-CoV-2 particles (MOI of 0.3) were first subjected to trypsin treatment for 15 min at 37°C and then exposed to buffers at the indicated pH for 10 min at 37°C, and vice versa. Calu-3 and Vero cells were then infected at a MOI of 0.3 and analyzed by flow cytometry as described in Fig. 1D. n = 4-8 biological replicates.


SARS - CoV - 2 was first treated with trypsin or thermolysin and then allowed to infect A549 * and Vero cells ( MOIs ~ 0 . 2 and 0 . 3 ) in the continuous presence of SB412515 ( cathepsin L inhibitor ) . Infected cells were quantified by flow cytometry as described in Fig . 1D , and data were normalized to samples where SB412515 had been omitted . n = 4 biological replicates .

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SARS-CoV-2 was first treated with trypsin or thermolysin and then allowed to infect A549* and Vero cells (MOIs ~0.2 and 0.3) in the continuous presence of SB412515 (cathepsin L inhibitor). Infected cells were quantified by flow cytometry as described in Fig. 1D, and data were normalized to samples where SB412515 had been omitted. n = 4 biological replicates.


( A ) Live - cell nanoscopy of HeLa cells . COX8A - SNAP was labeled with SNAP - cell SiR and imaged with STED nanoscopy .

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(A) Live-cell nanoscopy of HeLa cells. COX8A-SNAP was labeled with SNAP-cell SiR and imaged with STED nanoscopy.


( B ) 3D architecture of cristae in HeLa cells visualized by FIB - SEM . The OM and the IBM are together shown as a transparent surface . Cristae of two mitochondria are depicted in light and dark blue . A twisted crista is shown in grey .

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(B) 3D architecture of cristae in HeLa cells visualized by FIB-SEM. The OM and the IBM are together shown as a transparent surface. Cristae of two mitochondria are depicted in light and dark blue. A twisted crista is shown in grey.


( C ) Live - cell recording as in ( A ) . In addition , mitochondrial DNA was stained with PicoGreen and imaged by confocal laser scanning microscopy . Arrows mark sites where nucleoids but no lamellar cristae are present .

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(C) Live-cell recording as in (A). In addition, mitochondrial DNA was stained with PicoGreen and imaged by confocal laser scanning microscopy. Arrows mark sites where nucleoids but no lamellar cristae are present.


( D ) STED nanoscopy of mitochondria immunolabeled for Mic60 and dsDNA . Arrows mark sites where nucleoids are present .

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(D) STED nanoscopy of mitochondria immunolabeled for Mic60 and dsDNA. Arrows mark sites where nucleoids are present.


( E ) Dual - color STED nanoscopy of cells labeled for COX8A - SNAP and immunolabeled for Mic60 . Arrows mark Mic60 clusters in the absence of fully developed lamellar cristae .

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(E) Dual-color STED nanoscopy of cells labeled for COX8A-SNAP and immunolabeled for Mic60. Arrows mark Mic60 clusters in the absence of fully developed lamellar cristae.


( A ) Composition of MICOS in WT cells and MICOS mutants . Mic60 specific antibodies were used to pull - down Mic60 and interacting proteins from insolated mitochondria . * * Unspecific band , due to the cross reaction of the anti - Mic25 antibody with Mic19 .

(A) Composition of MICOS in WT cells and MICOS mutants. Mic60 specific antibodies were used to pull-down Mic60 and interacting proteins from insolated mitochondria. ** Unspecific band, due to the cross reaction of the anti-Mic25 antibody with Mic19.


( B ) Oxygen consumption rate ( OCR ) of WT cells and MICOS mutants as analyzed by Seahorse Analyzer . Basal and maximal OCR are shown relative to the WT . Error bars : SEM , n = 6 .

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(B) Oxygen consumption rate (OCR) of WT cells and MICOS mutants as analyzed by Seahorse Analyzer. Basal and maximal OCR are shown relative to the WT. Error bars: SEM, n=6.


( C ) Mitochondrial networks of WT and MICOS mutant cells . Cells were immunolabeled for TOM20 and visualized by confocal microscopy . The inset shows a magnification of the respective overview image . Scale bar : 10 µm . ( D ) Quantification of the mitochondrial networks as shown in ( C ) . The evaluation was performed manually in a blinded approach based on pre - defined morphology criteria . Average and SD of 3 independent biological replicates are shown ( > 170 cells per sample and replicate ) .

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(C) Mitochondrial networks of WT and MICOS mutant cells. Cells were immunolabeled for TOM20 and visualized by confocal microscopy. The inset shows a magnification of the respective overview image. Scale bar: 10 µm. (D) Quantification of the mitochondrial networks as shown in (C). The evaluation was performed manually in a blinded approach based on pre-defined morphology criteria. Average and SD of 3 independent biological replicates are shown (>170 cells per sample and replicate).


( A ) Representative TEM recordings of WT cells and MICOS - KO mutants , as indicated .

(A) Representative TEM recordings of WT cells and MICOS-KO mutants, as indicated.


( B ) Quantification of the overall cristae morphology on TEM recordings . Data information : n : number of mitochondrial

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(B) Quantification of the overall cristae morphology on TEM recordings. Data information: n: number of mitochondrial sections


( C ) Quantification of detailed morphology of CMs in MICOS mutants ( left ) . TEM recordings were assigned to 8 classes ( right ) . Data information : n : number of mitochondrial

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(C) Quantification of detailed morphology of CMs in MICOS mutants (left). TEM recordings were assigned to 8 classes (right). Data information: n: number of mitochondrial sections


( D ) CJ frequency on TEM recordings . The number of CJs was manually determined and normalized to the length of the OM . Samples were compared to WT by a One - way ANOVA test . Error bars : SEM . Data information : n : number of

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(D) CJ frequency on TEM recordings. The number of CJs was manually determined and normalized to the length of the OM. Samples were compared to WT by a One-way ANOVA test. Error bars: SEM. Data information: n: number of CJs