NIH - 3T3 were treated with TGFβ or mock for 48h reduced ( GSH ) oxidized ( GSSG ) glutathione were measured by LC - MS / MS . Shown is GSH / GSSG .

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

NIH-3T3 cells were treated with TGFβ or mock for 48h and reduced (GSH) and oxidized (GSSG) glutathione were measured by LC-MS/MS. Shown is the GSH/GSSG ratio.


NIH - 3T3 were treated with TGFβ for 48h or mock incubated with CM - H2DCFDA for last 30 min . ROS were measured by flow cytometry for CM - H2DCFDA . Values are relative to mock - treated .

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

NIH-3T3 cells were treated with TGFβ for 48h or mock and incubated with CM-H2DCFDA for the last 30 min. ROS were measured by flow cytometry for CM-H2DCFDA. Values are relative to mock-treated cells.


Comparison of acid representation in human collagens . x - axis denotes shown by single letter code .

Comparison of the amino acid representation in all human proteins and all collagens. The x-axis denotes individual amino acids shown by single letter code.


Glycine proline representation in acid hydrolysates of untreated NIH - 3T3 their secreted ECM , produced as in Figure 1d . were measured by GC - MS .

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

Glycine and proline representation in protein acid hydrolysates of untreated NIH-3T3 cells and their secreted ECM, produced as in Figure 1d. Amino acids were measured by GC-MS.


Tracing of [ U - 13C ] L - glutamine ( [ U - 13C ] Gln ) into indicated metabolites . NIH - 3T3 were treated with TGFβ ( 2 ng / mL for experiments ) or mock for 48h , medium was replaced ( including treatments ) with DMEM lacking L - glutamine supplemented with [ U - 13C ] Gln for last 8h . Metabolic steady state was reached at this time point ( see kinetic labeling curves in Figure EV3b ) . Metabolites were measured by LC - MS . left graph shows total pool normalized to packed volume ; right side shows labeling . αKG , alpha - ketoglutarate ; AU , arbitrary units .

O O O O 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

Tracing of [U-13C] L-glutamine ([U-13C] Gln) into indicated metabolites. NIH-3T3 cells were treated with TGFβ (2 ng/mL for all experiments) or mock for 48h, and the medium was replaced (including all treatments) with DMEM lacking L-glutamine and supplemented with [U-13C] Gln for the last 8h. Metabolic steady state was reached at this time point (see kinetic labeling curves in Figure EV3b). Metabolites were measured by LC-MS. The left graph shows the total pool size normalized to the packed cell volume; the right side shows the percent labeling. αKG, alpha-ketoglutarate; AU, arbitrary units.


Glutamine flux into glutamate , αKG proline in NIH - 3T3 treated with TGFβ or mock for 48h , relative to mock - treated .

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

Glutamine flux into glutamate, αKG and proline in NIH-3T3 cells treated with TGFβ or mock for 48h, relative to mock-treated cells.


Abundance of indicated metabolites in NIH - 3T3 treated with TGFβ or mock for 48h , relative to mock - treated . Metabolites were measured by GC - MS .

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

Abundance of indicated metabolites in NIH-3T3 cells treated with TGFβ or mock for 48h, relative to mock-treated cells. Metabolites were measured by GC-MS.


Representative MS2 spectra of CO1A1 peptide in ECM generated with unlabeled ( [ 12C ] Gln , left ) fully labeled glutamine ( [ U - 13C ] Gln , right ) . Peaks representing fully labeled ( m + 5 , m + 10 ) proline are highlighted in red . Peptides were measured by LC - MS .

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

Representative MS2 spectra of the CO1A1 peptide in ECM generated with unlabeled ([12C] Gln, left) and fully labeled glutamine ([U-13C] Gln, right). Peaks representing fully labeled (m+5, m+10) proline are highlighted in red. Peptides were measured by LC-MS.


Relative abundance of analytical peptide containing unlabeled ( m + 0 ) or one / two fully labelled prolines ( m + 5 / m + 10 ) from CO1A1 peptide in ECM derived from NIH - 3T3 treated with TGFβ or mock in presence of fully labelled L - glutamine ( [ U - 13C ] Gln ) . Peptides were measured by LC - MS . amount of ECM analyzed was normalized to packed volume of grown on a parallel plate under identical conditions .

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

Relative abundance of the analytical peptide containing unlabeled (m+0) or one/two fully labelled prolines (m+5/m+10) from the CO1A1 peptide in ECM derived from NIH-3T3 cells treated with TGFβ or mock in the presence of fully labelled L-glutamine ([U-13C] Gln). Peptides were measured by LC-MS. The amount of ECM analyzed was normalized to the packed cell volume of cells grown on a parallel plate under identical conditions.


Western blot of NIH - 3T3 treated with TGFβ ( 2 ng / mL for experiments ) or mock for 48h in presence of 0 . 5 % FBS .

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

Western blot of NIH-3T3 cells treated with TGFβ (2 ng/mL for all experiments) or mock for 48h in the presence of 0.5% FBS.


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

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 expressing sgCtrl or sgP5CS - 2 treated with TGFβ or mock for 48h in presence or absence of 0 . 15 mM proline .

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

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 expressing sgCtrl or sgP5CS - 2 grown in presence or absence of TGFβ 0 . 15 mM proline , measured by picrosirius red normalized to packed volume of grown on a parallel plate under identical conditions . Values are relative to mock - treated sgCtrl expressing .

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

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 expressing empty vector or HA - P5CS cDNA , measured by GC - MS . Values are relative to mock - treated empty vector expressing .

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

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 expressing empty vector or HA - P5CS cDNA .

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

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


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

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

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


of indicated gene expression datasets for mRNA 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 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

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 with idiopathic pulmonary fibrosis ( IPF ) compared to normal controls ( Ctrl ) . AU , arbitrary units . number of 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

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 forced vital capacity ( FVC ) before bronchodilator ( pre - BD ) as of what was predicted for each , from clinical data of GSE32537 .

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

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 expressing sgCtrl , sgSmad4 - 1 or sgSmad4 - 2 were treated with TGFβ or mock for 48h medium was replaced ( including treatments ) with DMEM without L - glutamine supplemented with [ U - 13C ] Gln for last 8h . Metabolites were measured by GC - MS .

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

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

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

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

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

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

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

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

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

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

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

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 B-CONTROLLED_VAR 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 were treated with TGFβ or mock for 1h , 6h , 24h or 48h .

O O O O O O O O O B-CONTROLLED_VAR 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 expressing sgCtrl , sgSmad4 - 1 or sgSmad4 - 2 were treated with TGFβ or mock for 6h , ROS were measured by flow cytometry for CM - H2DCFDA after incubation with CM - H2DCFDA for last 30 min of treatment . Values are normalized to mock - treated sgCtrl expressing .

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

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 expressing sgCtrl , sgSmad4 - 1 or sgSmad4 - 2 treated with TGFβ or mock for 6h in presence of 0 . 5 % FBS .

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

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 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 488nm lasers using flow cytometry . Oxidized roGFP2 gains excitability at 405nm loses excitability at 488nm . Oxidation status is expressed as of maximal oxidation which was determined by treating 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 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

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

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

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 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 experiments ) for 6h , ROS were measured by flow cytometry for CM - H2DCFDA after incubation with CM - H2DCFDA for last 30 min of treatment . Values are relative to control - treated . P - values represent comparison of samples to control .

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

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

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

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

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 expressing sgCtrl , sgSlc25a1 - 1 or sgSlc25a1 - 2 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 B-CONTROLLED_VAR 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 expressing sgCtrl , sgSlc25a1 - 1 or sgSlc25a1 - 2 grown in presence or absence of TGFβ , measured by picrosirius red normalized to packed volume of grown on a parallel plate under identical conditions . Values are relative to mock - treated sgCtrl expressing .

O 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

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

O O O O O O O B-CONTROLLED_VAR 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

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

O O O O O O O B-CONTROLLED_VAR 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

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 expressing empty vector or Slc25a1 cDNA treated with TGFβ or mock for 48h .

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

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 expressing empty vector or Slc25a1 cDNA grown in presence or absence of TGFβ , measured by picrosirius red normalized to packed volume of grown on a parallel plate under identical conditions . Values are relative to mock - treated empty vector expressing .

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

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.


of indicated gene expression datasets for mRNA 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 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

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);


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

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

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

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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.


( A ) Wild ‐ type ( YW5 ‐ 1B ) , apg10 ‐ 1 mutant ( MT91 ‐ 4 ‐ 2 ) Δapg10 ( TFD10 ‐ L1 ) were incubated in SD ( − N ) medium containing 1 mM PMSF at 30 ° C . After incubation for 6 h , were observed under microscopy ( Nomarski ) .

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(A) Wild‐type (YW5‐1B), apg10‐1 mutant (MT91‐4‐2) and Δapg10 (TFD10‐L1) cells were incubated in SD(−N) medium containing 1 mM PMSF at 30°C. After incubation for 6 h, cells were observed under light microscopy (Nomarski images).


( B ) Wild ‐ type ( closed circles ) , apg10 ‐ 1 mutant ( closed triangles ) Δapg10 ( open circles ) were grown to 1 OD600 / ml in YPD then transferred to SD ( − N ) medium . After incubation at 30 ° C for times indicated , their viability was determined by phloxine B ( Tsukada Ohsumi , 1993 ) .

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(B) Wild‐type (closed circles),apg10‐1 mutant (closed triangles) and Δapg10 (open circles) cells were grown to 1 OD600/ml in YPD and then transferred to SD(−N) medium. After incubation at 30°C for the times indicated, their viability was determined by phloxine B staining ( Tsukada and Ohsumi, 1993).


( C ) lysates from wild ‐ type with empty vector pRS316 ( lane 1 ) or HAAPG12 ( lane 2 ) , also apg10 ‐ 1 ( lane 3 ) Δapg10 with HAAPG12 ( lane 4 ) were subjected to Western blotting with anti ‐ HA antibody ( 16B12 ) .

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(C) Cell lysates from wild‐type cells with empty vector pRS316 (lane 1) or HAAPG12 (lane 2), and also apg10‐1 (lane 3) and Δapg10 cells with HAAPG12 (lane 4) were subjected to Western blotting analysis with anti‐HA antibody (16B12).


( A ) Two ‐ hybrid for interactions of Apg10p with Apg5p , Apg7p Apg12p . Yeast ( PJ69 ‐ 4A ) harboring indicated plasmids were streaked out on plates with medium lacking adenine to for interaction ‐ dependent activation of ADE2 gene .

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(A) Two‐hybrid assay for interactions of Apg10p with Apg5p, Apg7p and Apg12p. Yeast cells (PJ69‐4A) harboring the indicated plasmids were streaked out on plates with medium lacking adenine to assay for interaction‐dependent activation of the ADE2 gene.


( B ) Total lysates from Δapg5 with HAAPG10 / or MycAPG12 on 2μ plasmid were immunoprecipitated with anti ‐ HA ( mAb 16B12 ) or anti ‐ Myc ( mAb 9E10 ) antibody . Precipitates were analyzed by Western blotting using anti ‐ HA or anti ‐ Myc antibody . Apg12p adduct of Apg10p is indicated by ● an unidentified of 67 kDa by ● ● . positions of cross ‐ reacting IgG heavy chain ( H ) chain ( L ) are indicated by arrowheads .

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(B) Total lysates from Δapg5 cells with HAAPG10 and/or MycAPG12 on 2μ plasmid were immunoprecipitated with anti‐HA (mAb 16B12) or anti‐Myc (mAb 9E10) antibody. Precipitates were analyzed by Western blotting using anti‐HA or anti‐Myc antibody. Apg12p adduct of Apg10p is indicated by ● and an unidentified complex of 67 kDa by ●●. The positions of cross‐reacting IgG heavy chain (H) and light chain (L) are indicated by arrowheads.


Effects of deletion of APG5 or APG7 on Apg12p - Apg10p thioester formation . Coimmunoprecipitation was performed with wild ‐ type ( KA311B ) , Δapg5 ( YNM122 ) or Δapg7 ( YTS12 ) harboring HAAPG10 MycAPG12 on 2μ plasmids . resulting precipitates were analyzed by Western blotting . DTT ‐ resistant Apg12p - Apg5p conjugate is indicated by ● .

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Effects of the deletion of APG5 or APG7 on Apg12p-Apg10p thioester formation. Coimmunoprecipitation was performed with wild‐type (KA311B), Δapg5 (YNM122) or Δapg7 (YTS12) cells harboring HAAPG10 and MycAPG12 on 2μ plasmids. The resulting precipitates were analyzed by Western blotting. The DTT‐resistant Apg12p-Apg5p conjugate is indicated by ●.


( A ) lysates from Δapg10 ( TFD10 ‐ L1 ) harboring HAAPG12 each mutant form of HAAPG10 on CEN plasmids were subjected to Western blotting with anti ‐ HA antibody ( mAb 16B12 ) .

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(A) Cell lysates from Δapg10 cells (TFD10‐L1) harboring HAAPG12 and each mutant form of HAAPG10 on CEN plasmids were subjected to Western blotting analysis with anti‐HA antibody (mAb 16B12).


( B ) extracts from wild ‐ type ( KA311B ) harboring MycAPG12 mutated HAAPG10 were subjected to immunoprecipitation resulting precipitates were analyzed by Western blotting with rabbit anti ‐ HA antibody .

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(B) Cell extracts from the wild‐type cells (KA311B) harboring MycAPG12 and mutated HAAPG10 were subjected to immunoprecipitation and the resulting precipitates were analyzed by Western blotting with the rabbit anti‐HA antibody.


( A ) Autophagic activities were measured by an ALP ( Noda Ohsumi , 1998 ) . YTS3 ( Δapg10PHO8 : : pho8Δ60 ) harboring each mutant APG10 on CEN plasmids were grown to 1 OD600 / ml in SC medium lacking tryptophan then transferred to SD ( − N ) medium . Lysates from after incubation for 0 4 h were used for . Error bars indicate SD of three independent experiments .

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(A) Autophagic activities were measured by an ALP assay ( Noda and Ohsumi, 1998). YTS3 cells (Δapg10PHO8::pho8Δ60) harboring each mutant APG10 on CEN plasmids were grown to 1 OD600/ml in SC medium lacking tryptophan and then transferred to SD(−N) medium. Lysates from the cells after incubation for 0 and 4 h were used for assay. Error bars indicate the SD of three independent experiments.


( B ) lysates from Δapg10 ( TFD10 ‐ L1 ) harboring empty vector , pRS316 ( lane 1 ) , wild ‐ type HAAPG10 ( lane 2 ) , HAAPG10C26S ( lane 3 ) , HAAPG10C133S ( lane 4 ) or HAAPG10C137S ( lane 5 ) on CEN plasmids were subjected to Western blotting with anti ‐ API antiserum .

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(B) Cell lysates from Δapg10 cells (TFD10‐L1) harboring empty vector, pRS316 (lane 1), wild‐type HAAPG10 (lane 2), HAAPG10C26S (lane 3), HAAPG10C133S (lane 4) or HAAPG10C137S (lane 5) on CEN plasmids were subjected to Western blotting analysis with anti‐API antiserum.


Coronal axial brain MRI of with compound heterozygote mutations in ECE2 shows nodules of heterotopic neurons lining lateral ventricles .

Coronal and axial brain MRI of the patient with compound heterozygote mutations in ECE2 shows nodules of heterotopic neurons lining the lateral ventricles.


ECE2 expression on level . In situ hybridisation for ECE2 in 50d old cerebral organoids ( COs ) shows higher signal in cortical plate - like zone ( CP ' ) ( Scalebar = 100 µm ) . Ventricle - like lumen in COs is marked as V ' .

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ECE2 expression on RNA level. In situ hybridisation for ECE2 RNA in 50d old cerebral organoids (COs) shows higher signal in the cortical plate-like zone (CP') (Scalebar=100 µm). Ventricle-like lumen in COs is marked as V'.


ECE2 expression on level . Immunohistochemistry ( ) for ECE2 in 50d old COs shows accumulation in CP ' at apical surface ( Scalebar = 50µm ) . Ventricle - like lumen in COs is marked as V ' .

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ECE2 expression on protein level. Immunohistochemistry (IHC) for ECE2 in 50d old COs shows accumulation in the CP' and at the apical surface (Scalebar=50µm). Ventricle-like lumen in COs is marked as V'.


COs transfected with micro - RNAs targeting ECE2 ( KD ) or scrambled negative control ( CTRL ) GFP analysed 7 days later reveal an increase in ectopic neurons upon ECE2 KD ( Transfected are shown in green , NEUN + neuronal nuclei in magenta . d = day . Scalebar = 25 µm ) . Ventricle - like lumen in COs is marked as V ' .

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COs transfected with micro-RNAs targeting ECE2 (KD) or scrambled negative control (CTRL) and GFP and analysed 7 days later reveal an increase in ectopic neurons upon ECE2 KD (Transfected cells are shown in green, NEUN+ neuronal nuclei in magenta. d=day. Scalebar=25 µm). Ventricle-like lumen in COs is marked as V'.


Graph depicting number of ectopically located NEUN + in CTRL KD electroporated COs using two different micro - RNAs targeting ECE2 . Data shown as box plot ( mean = red , median = black , box represents 25th 75th percentiles , whiskers extend to 10th 90th percentiles , outliers are shown ; n = number of ventricles analysed ; * * * P < 0 . 001 in Kruskal - Wallis One Way ANOVA on Ranks Dunn ' s Pairwise Multiple Comparison ) .

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Graph depicting the number of ectopically located NEUN+ cells in CTRL and KD electroporated COs using two different micro-RNAs targeting ECE2. Data shown as box plot (mean = red line, median = black line, box represents 25th and 75th percentiles, whiskers extend to 10th and 90th percentiles, all outliers are shown; n=number of ventricles analysed; ***P < 0.001 in Kruskal-Wallis One Way ANOVA on Ranks and Dunn's Pairwise Multiple Comparison).


Scheme depicting experimental setup of neuronal differentiation timelapse imaging under treatment with ECE2 - inhibitor PHOS [ 42 ] .

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Scheme depicting the experimental setup of neuronal differentiation and timelapse imaging under treatment with the ECE2-inhibitor PHOS [42].


Graphs showing different parameters of neuronal migration dynamics , tracked via live imaging . Upon ECE2 inhibition , significant decrease in velocity , increase in resting TP increase in tortuosity are observed . Data shown as z - scores ( CTRL = DMSO control ; PHOS = Phosphoramidon ; Resting TP = Resting timepoints ; N = number of neurons analysed from 2 batches ; Resting TP : * P = 0 . 024 ; tortuosiy : * P = 0 . 035 , * * * P < 0 . 001 in 2 - Chi - Sqare ) .

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Graphs showing different parameters of neuronal migration dynamics, tracked via live imaging. Upon ECE2 inhibition, significant decrease in velocity, increase in resting TP and increase in tortuosity are observed. Data shown as z-scores (CTRL = DMSO control; PHOS = Phosphoramidon; Resting TP = Resting timepoints; N=number of individual neurons analysed from 2 batches; Resting TP: *P=0.024; tortuosiy: *P=0.035, ***P < 0.001 in 2-tailed Chi-Sqare test).


of distribution of GFP + transfected 1dpe upon CTRL Ece2 KD by IUE ( n = 3 CTRL 4 Ece2 KD brains ; * P = 0 . 044 in One - way ANOVA Tukey ' s Multiple Pairwise Comparison ; data shown as mean ± ) .

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Quantification of distribution of GFP+ transfected cells 1dpe upon CTRL and Ece2 KD by IUE (n=3 CTRL and 4 Ece2 KD brains; *P=0.044 in One-way ANOVA and Tukey's Multiple Pairwise Comparison; data shown as mean±SEM).


of GFP ( transfected ) Pax6 1 dpe showing ectopic Pax6 + progenitors ( red circle ) a reduction in their apical localisation ( yellow circle ) .

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IHC staining of GFP (transfected cells) and Pax6 1 dpe showing ectopic Pax6+ progenitors (red circle) and a reduction in their apical localisation (yellow circle).


High magnification of GFP Pax6 showing that ectopic progenitors are mostly not transfected . D - J , E13 - 16 Ece2 KD .

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High magnification images of GFP and Pax6 showing that ectopic progenitors are mostly not transfected. D-J, E13-16 Ece2 KD.


GFP - Pax6 + progenitors delaminate upon Ece2 KD ( red arrows ) , leaving behind a region free of Pax6 + in VZ ( yellow circle ) forming ectopic rosettes ( yellow box ) .

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GFP-Pax6+ progenitors delaminate upon Ece2 KD (red arrows), leaving behind a region free of Pax6+ cells in the VZ (yellow circle) and forming ectopic rosettes (yellow box).


Reduction in apical Arl13b in electroporation site ( arrowheads ) indicates loss of apical belt integrity disruption of apico - basal polarity upon Ece2 KD .

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Reduction in apical Arl13b in the electroporation site (white arrowheads) indicates loss of apical belt integrity and disruption of apico-basal polarity upon Ece2 KD.


Inside nodule , which is surrounded by thickened GFP + processes ( pink arrows ) , Arl13b shows formation of an ectopic apical

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Inside the nodule, which is surrounded by thickened GFP+ processes (pink arrows), Arl13b staining shows the formation of an ectopic apical surface


Reduction in β - catenin in electroporation site ( arrowheads ) indicates loss of apical belt integrity disruption of apico - basal polarity upon Ece2 KD .

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Reduction in β-catenin in the electroporation site (white arrowheads) indicates loss of apical belt integrity and disruption of apico-basal polarity upon Ece2 KD.


, Deep layer I , upper layer neurons localise next to ectopic rosettes , forming nodules of ectopic neurons ( blue arrowheads for single ectopic neurons blue circle for large nodule ) .

, Deep layer and I, upper layer neurons localise next to ectopic rosettes, forming nodules of ectopic neurons (blue arrowheads for single ectopic neurons and blue circle for large nodule).


at 3 dpe , counting proportion of brains with reduced density of Pax6 in VZ , ectopic progenitors or neurons , changes in apico - basal polarity ( including ectopic apical surface formation reduction in apically localised apical markers ; n = 6 brains per condition ; Exact binomial : * * * * P < 0 . 0001 ) . Scalebars in A , B , D , E , H , I = 100 µm ; in C , F , G Scalebar = 50 µm . V marks position of ventricle .

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Quantification at 3 dpe, counting the proportion of brains with reduced density of Pax6 in the VZ, ectopic progenitors or neurons, and changes in apico-basal polarity (including ectopic apical surface formation and reduction in apically localised apical markers; n=6 brains per condition; Exact binomial test: ****P < 0.0001). Scalebars in A, B, D, E, H, I=100 µm; in C, F, G Scalebar = 50 µm. V marks the position of the ventricle.


Brightfield of 60d old CTRL ECE2 KO COs show normal formation of neuroepithelial structures .

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Brightfield images of 60d old CTRL and ECE2 KO COs show normal formation of neuroepithelial structures.


ISH for ECE2 confirms absence of ECE2 mRNA in 60d old ECE2 KO COs ( pink dotted indicates position of CP ' ) .

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ISH for ECE2 confirms the absence of ECE2 mRNA in 60d old ECE2 KO COs (pink dotted line indicates position of the CP').


, FACS of 60d old ECE2 KO COs reveals an increase in progenitors decrease in neurons ( n = 7 - 8 samples of 3 pooled organoids each from 3 different batches ; * * P < 0 . 01 ; * * * P < 0 . 001 in One Way ANOVA Tukey Pairwise Multiple Comparison ) .

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, FACS analysis of 60d old ECE2 KO COs reveals an increase in progenitors and decrease in neurons (n=7-8 samples of 3 pooled organoids each from 3 different batches; **P < 0.01; ***P < 0.001 in One Way ANOVA and Tukey Pairwise Multiple Comparison).


Example of for PH3 + in 60d old CTRL ECE2 KO COs . of PH3 + cycling in 60d old COs , normalised to of apical surface of ventricle - like structures , shows an increase upon ECE2 KO ( Data shown as box plots with median as black , mean as red ; n = number of analysed ventricles from 2 batches of COs ; One Way ANOVA : * * P = 0 . 004 ) .

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Example images of IHC for PH3+ cells in 60d old CTRL and ECE2 KO COs. Quantification of PH3+ cycling cells in 60d old COs, normalised to length of the apical surface of ventricle-like structures, shows an increase upon ECE2 KO (Data shown as box plots with median as black line, mean as red line; n=number of analysed ventricles from 2 batches of COs; One Way ANOVA: **P = 0.004).


In ECE2 KO COs , more ventricles are disorganised in terms of neuronal localisation as revealed from MAP2 + bodies in progenitor zone ( n = number of ventricles from 2 independent batches ; two - Mann Whitney U ( U = 458 ) chi - square : χ2 ( 2 ) = 25 . 42 , * * * * P < 0 . 0001 ) .

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In ECE2 KO COs, more ventricles are disorganised in terms of neuronal localisation as revealed from MAP2+ cell bodies in the progenitor zone (n=number of ventricles from 2 independent batches; two-tailed Mann Whitney U (U = 458) and chi-square test: χ2(2)=25.42, ****P < 0.0001).


of actin from Westernblot of whole CO lysate reveals a reduction in ECE2 KO COs ( n = 4 independent batches of CTRL ECE2 KO COs with 3 pooled COs each sample ; * P = 0 . 021 in Kruskal - Wallis One Way of Variance on Ranks Dunn ' s Pairwise Comparison ) .

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Quantification of actin levels from Westernblot of whole CO lysate reveals a reduction in ECE2 KO COs (n=4 independent batches of CTRL and ECE2 KO COs with 3 pooled COs each sample; *P = 0.021 in Kruskal-Wallis One Way Analysis of Variance on Ranks and Dunn's Pairwise Comparison).


Fractionation of G - F - actin from CTRL ECE2 KO COs via Westernblot additionally reveals an increase in G - at expense F - actin upon ECE2 KO , suggesting a reduction in F - actin belt integrity ( n = 4 independent batches of CTRL ECE2 KO COs with 3 pooled COs each ; * P = 0 . 021 in Kruskal - Wallis One Way of Variance on Ranks Dunn ' s Pairwise Comparison ) .

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Fractionation of G- and F-actin from CTRL and ECE2 KO COs and analysis via Westernblot additionally reveals an increase in G- at the expense F-actin upon ECE2 KO, suggesting a reduction in F-actin belt integrity (n=4 independent batches of CTRL and ECE2 KO COs with 3 pooled COs each; *P = 0.021 in Kruskal-Wallis One Way Analysis of Variance on Ranks and Dunn's Pairwise Comparison).


thickness of apical F - actin belt is increased in ECE2 KO COs . C , Example of apical F - actin belt in 60d old CTRL ECE2 KO COs with F - actin labeled by AlexaFluor594 - conjugated Phalloidin ( Scalebar = 100 µm ) . D , of thickness of apical F - actin belt in Fiji [ 86 ] by measuring of F - actin dividing by of apical surface reveals significant increase in ECE2 KO COs ( Box plots : mean = red , median = black , box represents 25th 75th percentiles , whiskers extend to 10th 90th percentiles , outliers are shown ; n = number of analysed ventricles in 2 batches ; Kruskal - Wallis One Way ANOVA on Ranks Dunn ' s Pariwise Multiple Comparison : * * * P < 0 . 001 ) .

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The thickness of the apical F-actin belt is increased in ECE2 KO COs. C, Example images of apical F-actin belt in 60d old CTRL and ECE2 KO COs with F-actin labeled by AlexaFluor594-conjugated Phalloidin (Scalebar=100 µm). D, Quantification of the thickness of apical F-actin belt in Fiji [86] by measuring the area of F-actin and dividing by the length of apical surface reveals significant increase in ECE2 KO COs (Box plots: mean=red line, median=black line, box represents 25th and 75th percentiles, whiskers extend to 10th and 90th percentiles, all outliers are shown; n=number of analysed ventricles in 2 batches; Kruskal-Wallis One Way ANOVA on Ranks and Dunn's Pariwise Multiple Comparison: ***P < 0.001).


for ARL13B F - ACTIN in 60d old CTRL ECE2 KO COs shows disruption of honeycomb - like structure of apical adherens belt of apico - basal polarity in terms of apically localised cilium in ECE2 KO COs ( Scalebar = 10µm ) .

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IHC for ARL13B and F-ACTIN in 60d old CTRL and ECE2 KO COs shows disruption of honeycomb-like structure of the apical adherens belt and of apico-basal polarity in terms of apically localised primary cilium in ECE2 KO COs (Scalebar = 10µm).


microtubule cytoskeleton is changed in COs upon ECE2 KO . Example of ac - tub in CTRL vs . ECE2 KO COs ( Scalebar = 100 µm ) .

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The microtubule cytoskeleton is changed in COs upon ECE2 KO. Example images of ac-tub IHC in CTRL vs. ECE2 KO COs (Scalebar = 100 µm).


microtubule cytoskeleton is changed in COs upon ECE2 KO . of ac - tub in ventricles of ECE2 KO COs by measurement of mean grey value in Fiji shows significant reduction upon ECE2 KO ( n = number of analysed ventricles ; * P = 0 . 019 in One Way ANOVA Tukey Pairwise Multiple Comparison ) .

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The microtubule cytoskeleton is changed in COs upon ECE2 KO. Quantification of ac-tub in ventricles of ECE2 KO COs by measurement of the mean grey value in Fiji shows significant reduction upon ECE2 KO (n=number of analysed ventricles; *P = 0.019 in One Way ANOVA and Tukey Pairwise Multiple Comparison).


Apico - basal polarity is impaired upon ECE2 inhibition KO as visible in example of ARL13B in CTRL vs . KO COs ( H ; Scalebar = 100 µm ) . of germinal zones for normally high ( " 1 " ) vs . reduced ( " 0 " ) apical ARL13B reveals a reduction in proportion of normal ventricles in absence of ECE2 ( n = number of analysed ventricles from 2 batches ; * * * * P < 0 . 0001 in exact binomial ) .

O O O O O O 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 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 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

Apico-basal polarity is impaired upon ECE2 inhibition and KO as visible in example images of ARL13B IHC in CTRL vs. KO COs (H; Scalebar = 100 µm). Quantification of germinal zones for normally high ("1") vs. reduced ("0") apical ARL13B reveals a reduction in the proportion of normal ventricles in the absence of ECE2 (n=number of analysed ventricles from 2 batches; ****P < 0.0001 in exact binomial test).


of CTRL ECE2 KO COs 1 dpe with GAP43 - GFP reveals RG that delaminated ( arrowheads ) / or lost their bipolar ( red arrowheads ) in KO COs ( Scalebar = 25 µm ) .

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Analysis of CTRL and ECE2 KO COs 1 dpe with GAP43-GFP reveals RG that delaminated (white arrowheads) and/or lost their bipolar morphology (red arrowheads) in the KO COs (Scalebar = 25 µm).


Binning 1 dpe after forced exression of ECE2 in developing mouse cortex reveals ectopic positioning of Pax6 + GFP + neural progenitors with significantly reduced localisation to bin2 ( A ) increased localisation of Ctip2 + deep layer neurons to bin2 with reduction in bin5 ( B ) ( for binning strategy see Figure EV2B ; n = 3 CTRL 5 ECE2 OX brains E13 - 14 ; data shown as bars with mean ± ; * P < 0 . 05 * * P < 0 . 01 in Student ' s t - ) .

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Binning analysis 1 dpe after forced exression of ECE2 in the developing mouse cortex reveals ectopic positioning of Pax6+GFP+ neural progenitors with significantly reduced localisation to bin2 (A) and increased localisation of Ctip2+ deep layer neurons to bin2 with reduction in bin5 (B) (for binning strategy see Figure EV2B; n=3 CTRL and 5 ECE2 OX brains E13-14; data shown as bars with mean±SEM; *P < 0.05 and **P < 0.01 in Student's t-test).


Example E13 - 14 of for Pax6 Ctip2 upon CTRL ECE2 OX conditions ( Scalebar = 100 µm ; electroporated shown in green ) . Ventricle marked by V .

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Example images E13-14 of IHC for Pax6 and Ctip2 upon CTRL and ECE2 OX conditions (Scalebar = 100 µm; electroporated cells shown in green). Ventricle marked by V.


E13 - 16 of ECE2 OX in developing mouse cortex reveal ectopic positioning of Sox2 + ( D ) progenitors Ctip2 + deep - ( D ) Satb2 + upper layer neurons ( E ) ( Scalebars = 100 µm ; electroporated shown in green ) . F , at 3dpe , counting proportion of brains with normal vs . abnormal positioning of Sox2 + neural progenitors Ctip2 + Satb2 + neurons that of brains with intact vs . disrupted basal ( n = 5 CTRL 8 ECE2 OX brains ; exact binomial : * * * * P < 0 . 0001 ) . Ventricle marked by V .

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

E13-16 images and analysis of ECE2 OX in the developing mouse cortex reveal ectopic positioning of Sox2+ (D) progenitors and Ctip2+ deep- (D) and Satb2+ upper layer neurons (E) (Scalebars = 100 µm; electroporated cells shown in green). F, Quantification at 3dpe, counting the proportion of brains with normal vs. abnormal positioning of Sox2+ neural progenitors and Ctip2+ and Satb2+ neurons and that of brains with intact vs. disrupted basal membrane (n=5 CTRL and 8 ECE2 OX brains; exact binomial test: ****P < 0.0001). Ventricle marked by V.


ECE2 OX in COs ( electroporated shown in green ; Scalebars = 50 µm ; n = number of CO ventricles analysed from 2 batches ) . G , Example of COs 7 days after CTRL or ECE2 OX electroporation for PH3 + mitotic NEUN + neuronal nuclei . H , of PH3 + relative to electroporated apical surface reveals a reduction upon ECE2 OX in CTRL CO background ( * P = 0 . 046 in Kruskal - Wallis One Way of Variance on Ranks Dunn ' s Pairwise Comparison ) . I , of GFP + neurons as proportion of GFP + reveals an increase upon ECE2 OX in COs ( * P = 0 . 021 in Kruskal - Wallis One Way of Variance on Ranks Dunn ' s Pairwise Comparison ) . J , of ectopic neurons localising to VZ ' relative to electroporated radial units ( GFP + in VZ ' ) reveals a significant increase upon ECE2 OX ( * * * P < 0 . 001 in Kruskal - Wallis One Way of Variance on Ranks Dunn ' s Pairwise Comparison ) . Ventricle - like lumen marked by V ' .

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

ECE2 OX in COs (electroporated cells shown in green; Scalebars = 50 µm; n = number of CO ventricles analysed from 2 batches). G, Example images of COs 7 days after CTRL or ECE2 OX electroporation and IHC for PH3+ mitotic cells and NEUN+ neuronal nuclei. H, Quantification of PH3+ cells relative to electroporated apical surface length reveals a reduction upon ECE2 OX in CTRL CO background (*P = 0.046 in Kruskal-Wallis One Way Analysis of Variance on Ranks and Dunn's Pairwise Comparison). I, Quantification of GFP+ neurons as proportion of all GFP+ cells reveals an increase upon ECE2 OX in COs (*P = 0.021 in Kruskal-Wallis One Way Analysis of Variance on Ranks and Dunn's Pairwise Comparison). J, Quantification of ectopic neurons localising to the VZ' relative to electroporated radial units (GFP+ cells in the VZ') reveals a significant increase upon ECE2 OX (***P < 0.001 in Kruskal-Wallis One Way Analysis of Variance on Ranks and Dunn's Pairwise Comparison). Ventricle-like lumen marked by V'.


, Forced expression of ECE2 in ECE2 KO COs rescues neuronal positioning . K , Example of CTRL ECE2 electroporated ECE2 KO COs 7 dpe . L , of ectopic neurons relative to electroporated radial units reveals a decrease upon ECE2 OX in ECE2 KO COs , thus rescueing neuronal positioning phenotype in absence of ECE2 OX ( * * P = 0 . 002 in Kruskal - Wallis One Way of Variance on Ranks Dunn ' s Pairwise Comparison ) . Ventricle - like lumen marked by V ' .

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

, Forced expression of ECE2 in ECE2 KO COs rescues neuronal positioning. K, Example images of CTRL and ECE2 electroporated ECE2 KO COs 7 dpe. L, Quantification of ectopic neurons relative to electroporated radial units reveals a decrease upon ECE2 OX in ECE2 KO COs, thus rescueing the neuronal positioning phenotype in the absence of ECE2 OX (**P = 0.002 in Kruskal-Wallis One Way Analysis of Variance on Ranks and Dunn's Pairwise Comparison). Ventricle-like lumen marked by V'.


Volcano plot visualizes proteomic from 60d old CO lysates showing with significantly lower higher expression upon ECE2 KO ( n = 3 batches of 60d old CTRL ECE2 KO COs ; FDR 0 . 05 ; s0 1 ; dashed lines indicating cut - off ; names of example are shown ) .

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Volcano plot visualizes the proteomic analysis from 60d old CO lysates showing proteins with significantly lower and higher expression upon ECE2 KO (n=3 batches of 60d old CTRL and ECE2 KO COs; FDR 0.05; s0 1; dashed lines indicating the cut-off; names of example proteins are shown).


Volcano plot of proteomic of culture supernatant from 55d old CTRL ECE2 KO COs shows two significantly downregulated ( red ) in secretome ( n = 3 batches of CTRL ECE2 KO COs ; FDR 0 . 05 ; s0 1 ; dashed lines indicating cut - off ) . Non - significantly changed of interest are highlighted in green below cut - off curves .

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Volcano plot of proteomic analysis of cell culture supernatant from 55d old CTRL and ECE2 KO COs shows two significantly downregulated proteins (red) in the secretome (n=3 batches of CTRL and ECE2 KO COs; FDR 0.05; s0 1; dashed lines indicating the cut-off). Non-significantly changed proteins of interest are highlighted in green below the cut-off curves.


( B ) Representative emission spectra of mixtures containing ECFP - D4H , mVenus - Lact - C2 , liposomes containing indicated compositions of lipids . Note that FRET signal was increased in presence of both cholesterol ( 50 % ) phosphatidylserine ( DOPS ) ( 20 % ) that FRET signal was decreased when sphingomyelin ( SM ) ( 25 % ) was additionally incorporated into liposomes . Asterisks indicate positions of emission maximum of ECFP ( * 477 nm ) FRET ( * * 525 nm ) . DOPC , phosphatidylcholine ( 1 , 2 - dioleoyl - sn - glycero - 3 - phosphocholine ) ; DOPS , phosphatidylserine ( 1 , 2 - dioleoyl - sn - glycero - 3 - phospho - L - serine ) ; Chol . , cholesterol ; SM , sphingomyelin ( N - oleoyl - D - erythro - sphingosylphosphorylcholine ) . ( C ) of of FRET signal at 525 nm to ECFP emission at 480 nm ( FRET / ECFP ) ( see Materials Methods ) from mixtures as shown in ( D ) ( mean ± , n = 4 independent experiments ; Dunnett ' s multiple comparisons , * * P < 0 . 0001 ) .

O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Representative emission spectra of mixtures containing ECFP-D4H, mVenus-Lact-C2, and liposomes containing indicated compositions of lipids. Note that FRET signal was increased in the presence of both cholesterol (50%) and phosphatidylserine (DOPS) (20%) and that FRET signal was decreased when sphingomyelin (SM) (25%) was additionally incorporated into the liposomes. Asterisks indicate positions of emission maximum of ECFP (*477 nm) and FRET (**525 nm). DOPC, phosphatidylcholine (1,2-dioleoyl-sn-glycero-3-phosphocholine); DOPS, phosphatidylserine (1,2-dioleoyl-sn-glycero-3-phospho-L-serine); Chol., cholesterol; SM, sphingomyelin (N-oleoyl-D-erythro-sphingosylphosphorylcholine). (C) Quantification of the ratio of FRET signal at 525 nm to ECFP emission at 480 nm (FRET/ECFP) (see Materials and Methods) from mixtures as shown in (D) (mean ± SEM, n = 4 independent experiments; Dunnett's multiple comparisons test, **P < 0.0001).


( D ) Left : Time course of normalized mCherry signal , as assessed by TIRF microscopy , from GRAMD1 triple knockout ( TKO ) HeLa expressing mCherry - GRAM1b . were pre - incubated with either control imaging buffer ( for control ) or imaging buffer containing purified ECFP - D4H ( 3 µM ) ( for masking accessible PM cholesterol ) for 30 min at 37 ° C imaged with same buffer conditions . SMase treatment ( 100 mU / ml ) is indicated . Right : Values of ΔF / F0 corresponding to end of experiment as indicated by arrow [ mean ± , n = 32 ( Control ) , n = 35 ( Pre - incubation w / ECFP - D4H ) ; data are pooled from two independent experiments for each condition ; two - unpaired Student ' s t - , * * P < 0 . 0001 ] .

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(D) Left: Time course of normalized mCherry signal, as assessed by TIRF microscopy, from GRAMD1 triple knockout (TKO) HeLa cells expressing mCherry-GRAM1b. Cells were pre-incubated with either control imaging buffer (for control) or imaging buffer containing purified ECFP-D4H proteins (3 µM) (for masking accessible PM cholesterol) for 30 min at 37°C and imaged with the same buffer conditions. SMase treatment (100 mU/ml) is indicated. Right: Values of ΔF/F0 corresponding to the end of the experiment as indicated by the arrow [mean ± SEM, n = 32 cells (Control), n = 35 cells (Pre-incubation w/ ECFP-D4H); data are pooled from two independent experiments for each condition; two-tailed unpaired Student's t-test, **P < 0.0001].


( E ) Left : Time course of normalized EGFP signal , as assessed by TIRF microscopy , from GRAMD1 triple knockout ( TKO ) HeLa expressing EGFP - GRAM1b together with either mCherry for control or mCherry - Lact - C2 for PS masking . SMase treatment ( 100 mU / ml ) is indicated . Right : Values of ΔF / F0 corresponding to end of experiment as indicated by arrow [ mean ± , n = 53 ( mCherry ) , n = 32 ( mCherry - Lact - C2 OE ) ; data are pooled from four independent experiments for each condition ; two - unpaired Student ' s t - , * * P < 0 . 0001 ] .

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(E) Left: Time course of normalized EGFP signal, as assessed by TIRF microscopy, from GRAMD1 triple knockout (TKO) HeLa cells expressing EGFP-GRAM1b together with either mCherry for control or mCherry-Lact-C2 for PS masking. SMase treatment (100 mU/ml) is indicated. Right: Values of ΔF/F0 corresponding to the end of the experiment as indicated by the arrow [mean ± SEM, n = 53 cells (mCherry), n = 32 cells (mCherry-Lact-C2 OE); data are pooled from four independent experiments for each condition; two-tailed unpaired Student's t-test, **P < 0.0001].


( C - E ) Liposome sedimentation assays of wild - type GRAM1b ( WT ) mutant versions of GRAM1b carrying indicated mutations ( K161A , R191A , K161A / R191A ) . Liposomes containing indicated mole % lipids were incubated with purified GRAM1b as shown . Bound [ pellet , ( P ) ] were separated from unbound [ supernatant , ( S ) ] , run on SDS - PAGE visualized by colloidal blue ( mean ± , n = 3 independent experiments for conditions in phosphatidylserine ( PS ) sensitivity , co - incidence detection , anionic lipid selectivity assays ; n = 12 independent experiments for WT , n = 3 independent experiments for K161A R191A , n = 6 independent experiments for K161A / R191A in cholesterol sensitivity ) . DOPC , phosphatidylcholine ( 1 , 2 - dioleoyl - sn - glycero - 3 - phosphocholine ) ; DOPS , phosphatidylserine ( 1 , 2 - dioleoyl - sn - glycero - 3 - phospho - L - serine ) ; Chol . , cholesterol ; PA , phosphatidic acid ( 1 , 2 - dioleoyl - sn - glycero - 3 - phosphate ) ; PI ( 4 ) P , phosphatidylinositol 4 - phosphate ; PI ( 4 , 5 ) P2 , phosphatidylinositol 4 , 5 - bisphosphate ; PI , L - α - phosphatidylinositol .

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(C-E) Liposome sedimentation assays of wild-type GRAM1b proteins (WT) and mutant versions of GRAM1b proteins carrying indicated mutations (K161A, R191A, and K161A/R191A). Liposomes containing the indicated mole% lipids were incubated with purified GRAM1b proteins as shown. Bound proteins [pellet, (P)] were separated from the unbound proteins [supernatant, (S)], run on SDS-PAGE and visualized by colloidal blue staining (mean ± SEM, n = 3 independent experiments for all conditions in phosphatidylserine (PS) sensitivity, co-incidence detection, and anionic lipid selectivity assays; n = 12 independent experiments for WT, n = 3 independent experiments for K161A and R191A, and n = 6 independent experiments for K161A/R191A in the cholesterol sensitivity assay). DOPC, phosphatidylcholine (1,2-dioleoyl-sn-glycero-3-phosphocholine); DOPS, phosphatidylserine (1,2-dioleoyl-sn-glycero-3-phospho-L-serine); Chol., cholesterol; PA, phosphatidic acid (1,2-dioleoyl-sn-glycero-3-phosphate); PI(4)P, phosphatidylinositol 4-phosphate; PI(4,5)P2, phosphatidylinositol 4,5-bisphosphate; PI, L-α-phosphatidylinositol.


( B ) Sterol specificity of GRAM domain of GRAMD1b ( GRAM1b ) as assessed via liposome sedimentation assays . Liposomes containing indicated mole % lipids were incubated with purified GRAM1b as indicated . Bound [ pellet , ( P ) ] were separated from unbound [ supernatant , ( S ) ] , run on SDS - PAGE visualized by colloidal blue ( mean ± , n = 6 independent experiments for liposomes containing only DOPC , n = 3 independent experiments for ) . DOPC , phosphatidylcholine ( 1 , 2 - dioleoyl - sn - glycero - 3 - phosphocholine ) .

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(B) Sterol specificity of the GRAM domain of GRAMD1b (GRAM1b) as assessed via liposome sedimentation assays. Liposomes containing the indicated mole% lipids were incubated with purified GRAM1b proteins as indicated. Bound proteins [pellet, (P)] were separated from the unbound proteins [supernatant, (S)], run on SDS-PAGE and visualized by colloidal blue staining (mean ± SEM, n = 6 independent experiments for liposomes containing only DOPC, n = 3 independent experiments for the rest). DOPC, phosphatidylcholine (1,2-dioleoyl-sn-glycero-3-phosphocholine).


( D - E ) Liposome sedimentation assays of wild - type GRAM1b ( WT ) mutant versions of GRAM1b carrying indicated mutations ( R189W , R189W / R191A ) . Liposomes containing indicated mole % lipids were incubated with purified GRAM1b as shown . Bound [ pellet , ( P ) ] were separated from unbound [ supernatant , ( S ) ] , run on SDS - PAGE visualized by colloidal blue [ mean ± , n = 3 independent experiments for conditions in co - incidence detection ; n = 3 independent experiments for R189W R189W / R191A , n = 6 independent experiments for WT in phosphatidylserine ( PS ) sensitivity ; n = 3 independent experiments for R189W R189W / R191A , n = 5 independent experiments for WT in cholesterol sensitivity ] . DOPC , phosphatidylcholine ( 1 , 2 - dioleoyl - sn - glycero - 3 - phosphocholine ) ; DOPS , phosphatidylserine ( 1 , 2 - dioleoyl - sn - glycero - 3 - phospho - L - serine ) ; Chol . , cholesterol .

O O O O O O O O O O O O B-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 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(D-E) Liposome sedimentation assays of wild-type GRAM1b proteins (WT) and mutant versions of GRAM1b proteins carrying indicated mutations (R189W, R189W/R191A). Liposomes containing the indicated mole% lipids were incubated with purified GRAM1b proteins as shown. Bound proteins [pellet, (P)] were separated from the unbound proteins [supernatant, (S)], run on SDS-PAGE and visualized by colloidal blue staining [mean ± SEM, n = 3 independent experiments for all conditions in the co-incidence detection assay; n = 3 independent experiments for R189W and R189W/R191A, n = 6 independent experiments for WT in phosphatidylserine (PS) sensitivity assay; n = 3 independent experiments for R189W and R189W/R191A, n = 5 independent experiments for WT in cholesterol sensitivity assay]. DOPC, phosphatidylcholine (1,2-dioleoyl-sn-glycero-3-phosphocholine); DOPS, phosphatidylserine (1,2-dioleoyl-sn-glycero-3-phospho-L-serine); Chol., cholesterol.


( F ) Confocal of live GRAMD1 TKO HeLa expressing either wild - type ( WT ) or mutant versions of EGFP - tagged GRAM domain of GRAMD1b ( EGFP - GRAM1b ) constructs as indicated . were treated with SMase ( 100 mU / ml for 1 hour at 37 ° C ) before imaging . Insets show at higher magnification regions indicated by dashed boxes . Anionic lipid sensing defective mutants ( K161A , R191A , K161A / R191A ) , a cholesterol sensing defective mutant ( R189W ) , a cholesterol / anionic lipid sensing defective mutant ( R189W / R191A ) are shown . Note absence of ( R189W , R189W / R191A , K161A / R191A ) or significantly reduced ( K161A , R191A ) PM recruitment of mutant versions of EGFP - GRAM1b compared to strong PM recruitment of wild - type EGFP - GRAM1b ( WT ) . Scale bars , 10 µm .

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(F) Confocal images of live GRAMD1 TKO HeLa cells expressing either wild-type (WT) or mutant versions of EGFP-tagged GRAM domain of GRAMD1b (EGFP-GRAM1b) constructs as indicated. Cells were treated with SMase (100 mU/ml for 1 hour at 37°C) before imaging. Insets show at higher magnification the regions indicated by white dashed boxes. Anionic lipid sensing defective mutants (K161A, R191A, and K161A/R191A), a cholesterol sensing defective mutant (R189W), and a cholesterol/anionic lipid sensing defective mutant (R189W/R191A) are shown. Note the absence of (R189W, R189W/R191A, and K161A/R191A) or significantly reduced (K161A, R191A) PM recruitment of the mutant versions of EGFP-GRAM1b compared to the strong PM recruitment of wild-type EGFP-GRAM1b (WT). Scale bars, 10 µm.


( G ) of of PM EGFP - GRAM1b signals to EGFP - GRAM1b signals , as assessed by confocal microscopy scan from GRAMD1 TKO HeLa expressing either wild - type ( WT ) or mutant versions of EGFP - GRAM1b with or without SMase treatment ( 100 mU / ml for 1 hour at 37 ° C ) as shown in ( F ) Appendix Fig S1B ( mean ± , n = 40 for WT , n = 30 for other conditions ; data are pooled from four independent experiments for WT three independent experiments for ; two - unpaired Student ' s t - , * * P < 0 . 0001 . n . s . denotes not significant ) .

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 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

(G) Quantification of the ratio of PM EGFP-GRAM1b signals to the cytosolic EGFP-GRAM1b signals, as assessed by confocal microscopy and line scan analysis from GRAMD1 TKO HeLa cells expressing either wild-type (WT) or mutant versions of EGFP-GRAM1b with or without SMase treatment (100 mU/ml for 1 hour at 37°C) as shown in (F) and Appendix Fig S1B (mean ± SEM, n = 40 cells for WT, and n = 30 cells for other conditions; data are pooled from four independent experiments for WT and three independent experiments for the rest; two-tailed unpaired Student's t-test, **P < 0.0001. n.s. denotes not significant).


( H ) Left : Time course of normalized EGFP signal , as assessed by TIRF microscopy , from GRAMD1 TKO HeLa expressing either wild - type ( WT ) or mutant versions of EGFP - GRAM1b constructs as indicated . SMase treatment ( 100 mU / ml ) is indicated . Right : Values of ΔF / F0 corresponding to end of experiment as indicated by arrow [ mean ± , n = 126 ( WT ) , n = 38 ( K161A ) , n = 33 ( R191A ) , n = 43 ( K161A / R191A ) , n = 46 ( R189W ) , n = 44 ( R189W / R191A ) ; data are pooled from six independent experiments for WT two independent experiments for ; Dunnett ' s multiple comparisons , * * P = 0 . 0002 ( WT vs . K161A ) * * P < 0 . 0001 for ] .

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

(H) Left: Time course of normalized EGFP signal, as assessed by TIRF microscopy, from GRAMD1 TKO HeLa cells expressing either wild-type (WT) or mutant versions of EGFP-GRAM1b constructs as indicated. SMase treatment (100 mU/ml) is indicated. Right: Values of ΔF/F0 corresponding to the end of the experiment as indicated by the arrow [mean ± SEM, n = 126 cells (WT), n = 38 cells (K161A), n = 33 cells (R191A), n = 43 cells (K161A/R191A), n = 46 cells (R189W), n = 44 cells (R189W/R191A); data are pooled from six independent experiments for WT and two independent experiments for the rest; Dunnett's multiple comparisons test, **P = 0.0002 (WT vs. K161A) and **P < 0.0001 for the rest].


( B ) Liposome tethering by purified GRAMD1b . Left : Representative time course of liposome tethering , as assessed by turbidity of mixtures containing both LPM LER together with either wild - type GRAMD1b ( WT ) , mutant GRAMD1b R189W ( R189W ) , mutant GRAMD1b R189W / R191A ( RW / RA ) , or buffer alone as indicated . Turbidity , as assessed by measuring absorbance values at 405 nm , reflects clustering of liposomes due to tethering of LPM LER . Right : Values of turbidity corresponding to end of experiment as indicated by arrow ( mean ± , n = 6 independent experiments for WT , n = 3 independent experiments for buffer alone , R189W , RW / RA ; Dunnett ' s multiple comparisons , * * P < 0 . 0001 )

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 B-MEASURED_VAR O O O O O B-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Liposome tethering by purified GRAMD1b proteins. Left: Representative time course of liposome tethering, as assessed by turbidity of mixtures containing both LPM and LER together with either wild-type GRAMD1b proteins (WT), mutant GRAMD1b R189W proteins (R189W), mutant GRAMD1b R189W/R191A proteins (RW/RA), or buffer alone as indicated. Turbidity, as assessed by measuring absorbance values at 405 nm, reflects clustering of liposomes due to tethering of LPM and LER. Right: Values of turbidity corresponding to the end of the experiment as indicated by the arrow (mean ± SEM, n = 6 independent experiments for WT, n = 3 independent experiments for buffer alone, R189W, and RW/RA; Dunnett's multiple comparisons test, **P < 0.0001)


( C ) DHE transfer between liposomes by purified GRAMD1b . Left : Representative time course of DHE transfer from LPM to LER mediated by either wild - type GRAMD1b ( WT ) , mutant GRAMD1b R189W ( R189W ) , mutant GRAMD1b R189W / R191A ( RW / RA ) , or buffer alone as indicated . Methyl - β - cyclodextrin ( MCD ) at 1 mM was used to determine DHE equilibration . Transfer of DHE from LPM to LER resulted in a decrease in resonance energy transfer ( FRET ) between DHE DNS - PE in LPM . A series of liposomes with different DHE mol % were prepared to plot a calibration curve to convert FRET signals to DHE molecules transferred ( see Appendix Fig S2D Materials Methods ) . Right : Values of DHE transfer rate of GRAMD1b ( WT ) , GRAMD1b ( R189W ) , GRAMD1b ( RW / RA ) , as estimated by FRET - lipid transfer [ mean ± , n = 6 independent experiments for WT , n = 3 independent experiments for R189W RW /

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

(C) DHE transfer between liposomes by purified GRAMD1b proteins. Left: Representative time course of DHE transfer from LPM to LER mediated by either wild-type GRAMD1b proteins (WT), mutant GRAMD1b R189W proteins (R189W), mutant GRAMD1b R189W/R191A proteins (RW/RA), or buffer alone as indicated. Methyl-β-cyclodextrin (MCD) at 1 mM was used to determine DHE equilibration. Transfer of DHE from LPM to LER resulted in a decrease in fluorescence resonance energy transfer (FRET) between DHE and DNS-PE in LPM. A series of liposomes with different DHE mol% were prepared to plot a calibration curve to convert FRET signals to DHE molecules transferred (see Appendix Fig S2D and Materials and Methods). Right: Values of DHE transfer rate of GRAMD1b (WT), GRAMD1b (R189W), and GRAMD1b (RW/RA), as estimated by the FRET-based lipid transfer assay [mean ± SEM, n = 6 independent experiments for WT, n = 3 independent experiments for R189W and RW/RA


( A ) Lysates of wild - type ( control ) GRAMD1 TKO ( TKO ) HeLa that stably expressed either EGFP or EGFP - GRAMD1b constructs as indicated [ wild - type ( WT ) , R189W mutant ( R189W ) , R189W / R191A mutant ( RW / RA ) ] were processed by SDS - PAGE immunoblotted ( IB ) with anti - GFP , anti - GRAMD1b , anti - actin antibodies .

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

(A) Lysates of wild-type (control) and GRAMD1 TKO (TKO) HeLa cells that stably expressed either EGFP or EGFP-GRAMD1b constructs as indicated [wild-type (WT), R189W mutant (R189W), R189W/R191A mutant (RW/RA)] were processed by SDS-PAGE and immunoblotted (IB) with anti-GFP, anti-GRAMD1b, and anti-actin antibodies.


( B ) Confocal of live GRAMD1 TKO HeLa stably expressing EGFP - GRAMD1b constructs as indicated that were additionally transfected with an accessible PM cholesterol biosensor , mCherry - tagged GRAM domain of GRAMD1b ( mCherry - GRAM1b ) . were treated with SMase ( 100 mU / ml for 1 hour at 37 ° C ) before imaging . Insets show at higher magnification regions indicated by dashed boxes . Note very weak PM recruitment of mCherry - GRAM1b in that stably expressed EGFP - GRAMD1b WT , compared to strong PM recruitment of mCherry - GRAM1b in that stably expressed EGFP - GRAMD1b R189W mutant ( R189W ) or EGFP - GRAMD1b R189W / R191A mutant ( RW / RA ) . Scale bars , 10 µm .

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

(B) Confocal images of live GRAMD1 TKO HeLa cells stably expressing EGFP-GRAMD1b constructs as indicated that were additionally transfected with an accessible PM cholesterol biosensor, mCherry-tagged GRAM domain of GRAMD1b (mCherry-GRAM1b). Cells were treated with SMase (100 mU/ml for 1 hour at 37°C) before imaging. Insets show at higher magnification the regions indicated by white dashed boxes. Note the very weak PM recruitment of mCherry-GRAM1b in cells that stably expressed EGFP-GRAMD1b WT, compared to the strong PM recruitment of mCherry-GRAM1b in cells that stably expressed EGFP-GRAMD1b R189W mutant (R189W) or EGFP-GRAMD1b R189W/R191A mutant (RW/RA). Scale bars, 10 µm.


( C ) of of PM mCherry - GRAM1b signals to mCherry - GRAM1b signals , as assessed by confocal microscopy scan from GRAMD1 TKO HeLa expressing mCherry - GRAM1b with SMase treatment ( 100 mU / ml for 1 hour at 37 ° C ) as shown in ( B ) Fig EV3B ( mean ± , n = 20 for each condition ; data are pooled from two independent experiments ; Dunnett ' s multiple comparisons , * * P < 0 . 0001 . n . s . denotes not significant ) .

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 B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Quantification of the ratio of PM mCherry-GRAM1b signals to the cytosolic mCherry-GRAM1b signals, as assessed by confocal microscopy and line scan analysis from GRAMD1 TKO HeLa cells expressing mCherry-GRAM1b with SMase treatment (100 mU/ml for 1 hour at 37°C) as shown in (B) and Fig EV3B (mean ± SEM, n = 20 cells for each condition; data are pooled from two independent experiments; Dunnett's multiple comparisons test, **P < 0.0001. n.s. denotes not significant).


( D ) Left : Time course of normalized mCherry signal , as assessed by TIRF microscopy , from GRAMD1 TKO HeLa stably expressing either EGFP or EGFP - GRAMD1b constructs as indicated that were additionally transfected with an accessible PM cholesterol biosensor mCherry - GRAM1b . SMase treatment ( 100 mU / ml ) is indicated . Right : Values of ΔF / F0 corresponding to end of experiment as indicated by arrow [ mean ± , n = 31 ( EGFP ) , n = 28 ( EGFP - GRAMD1b WT ) , n = 23 ( EGFP - GRAMD1b R189W ) , n = 27 [ EGFP - GRAMD1b R189W / R191A ( RW / RA ) ] ; data are pooled from two independent experiments for each condition ; Dunnett ' s multiple comparisons , * * P < 0 . 0001 ( EGFP vs . WT WT vs . R189W ) , * * P = 0 . 0002 ( WT vs . RW / RA ) ] .

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

(D) Left: Time course of normalized mCherry signal, as assessed by TIRF microscopy, from GRAMD1 TKO HeLa cells stably expressing either EGFP or EGFP-GRAMD1b constructs as indicated that were additionally transfected with an accessible PM cholesterol biosensor mCherry-GRAM1b. SMase treatment (100 mU/ml) is indicated. Right: Values of ΔF/F0 corresponding to the end of the experiment as indicated by the arrow [mean ± SEM, n = 31 cells (EGFP), n = 28 cells (EGFP-GRAMD1b WT), n = 23 cells (EGFP-GRAMD1b R189W), n = 27 cells [EGFP-GRAMD1b R189W/R191A (RW/RA)]; data are pooled from two independent experiments for each condition; Dunnett's multiple comparisons test, **P < 0.0001 (EGFP vs. WT and WT vs. R189W), **P = 0.0002 (WT vs. RW/RA)].


( E ) Wild - type ( control ) GRAMD1 TKO ( TKO ) HeLa that stably expressed either EGFP or EGFP - tagged GRAMD1b constructs as indicated [ wild - type ( WT ) , R189W mutant ( R189W ) , R189W / R191A mutant ( RW / RA ) ] , were cultured in medium supplemented with 10 % lipoprotein - deficient serum ( LPDS ) mevastatin ( 50 µM ) for 16 hours then treated with SMase ( 100 mU / ml ) for 3 hours at 37 ° C . Top : Lysates of were processed for SDS - PAGE IB with anti - SREBP - 2 anti - actin antibodies . Arrows indicate precursor ( P ) ( C ) forms of SREBP - 2 . Bottom : response rate was obtained by normalizing of band of SREBP - 2 over total band of precursor forms of SREBP - 2 from with SMase treatment by one from without SMase treatment for each condition . Note that suppression of SREBP - 2 cleavage is attenuated in GRAMD1 TKO HeLa compared to wild - type control HeLa . Note also rescue by expression of wild - type EGFP - GRAMD1b ( WT ) but not by mutant versions of EGFP - GRAMD1b [ R189W , R189W / R191A ( RW / RA ) ] [ mean ± , n = 4 lysates ( independent experiments ) for each condition ; Dunnett ' s multiple comparisons , * * P = 0 . 0035 ( Control + EGFP vs . TKO + EGFP ) , * * P < 0 . 0001 ( TKO + EGFP vs . TKO + WT ) , n . s . denotes not significant ] .

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

(E) Wild-type (control) and GRAMD1 TKO (TKO) HeLa cells that stably expressed either EGFP or EGFP-tagged GRAMD1b constructs as indicated [wild-type (WT), R189W mutant (R189W), R189W/R191A mutant (RW/RA)], were cultured in the medium supplemented with 10% lipoprotein-deficient serum (LPDS) and mevastatin (50 µM) for 16 hours and then treated with SMase (100 mU/ml) for 3 hours at 37°C. Top: Lysates of the cells were processed for SDS-PAGE and IB with anti-SREBP-2 and anti-actin antibodies. Arrows indicate precursor (P) and cleaved (C) forms of SREBP-2. Bottom: The response rate was obtained by normalizing the ratio of the band intensity of the cleaved SREBP-2 over the total band intensity of cleaved and precursor forms of SREBP-2 from the cells with SMase treatment by the one from the cells without SMase treatment for each condition. Note that the suppression of SREBP-2 cleavage is attenuated in GRAMD1 TKO HeLa cells compared to wild-type control HeLa cells. Note also the rescue by expression of wild-type EGFP-GRAMD1b (WT) but not by mutant versions of EGFP-GRAMD1b [R189W, R189W/R191A (RW/RA)] [mean ± SEM, n = 4 lysates (independent experiments) for each condition; Dunnett's multiple comparisons test, **P = 0.0035 (Control + EGFP vs. TKO + EGFP), **P < 0.0001 (TKO + EGFP vs. TKO + WT), n.s. denotes not significant].


( F ) Amphotericin B resistance of SMase - treated wild - type ( control ) GRAMD1 TKO ( TKO ) HeLa that stably expressed either EGFP or EGFP - tagged GRAMD1b constructs as indicated [ wild - type ( WT ) , R189W mutant ( R189W ) , R189W / R191A mutant ( RW / RA ) ] . Left : that had been pre - treated with SMase ( 100 mU / ml ) for 3 hours at 37 ° C were treated with indicated of Amphotericin B for 20 min at 37ºC . After overnight recovery in culture media , viability was measured by detecting ATP present in each well via luminescence ( see Materials Methods ) . same number of were seeded in each well before SMase treatment . Note reduced viability of with increasing amount of Amphotericin B . Right : of viability with increasing amount of Amphotericin B . Note resistance of wild - type control GRAMD1 TKO that stably expressed EGFP - GRAMD1b ( WT ) compared to GRAMD1 TKO or GRAMD1 TKO that stably expressed mutant versions of EGFP - GRAMD1b [ R189W , R189W / R191A ( RW / RA ) ] ( mean ± , n = 3 independent experiments for each condition ) .

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

(F) Amphotericin B resistance of SMase-treated wild-type (control) and GRAMD1 TKO (TKO) HeLa cells that stably expressed either EGFP or EGFP-tagged GRAMD1b constructs as indicated [wild-type (WT), R189W mutant (R189W), R189W/R191A mutant (RW/RA)]. Left: Cells that had been pre-treated with SMase (100 mU/ml) for 3 hours at 37°C were treated with indicated concentration of Amphotericin B for 20 min at 37ºC. After overnight recovery in culture media, cell viability was measured by detecting ATP present in each well via luminescence (see Materials and Methods). The same number of cells were seeded in each well before SMase treatment. Note the reduced viability of cells with increasing amount of Amphotericin B. Right: Quantification of cell viability with increasing amount of Amphotericin B. Note the resistance of wild-type control and GRAMD1 TKO cells that stably expressed EGFP-GRAMD1b (WT) compared to GRAMD1 TKO cells or GRAMD1 TKO cells that stably expressed mutant versions of EGFP-GRAMD1b [R189W, R189W/R191A (RW/RA)] (mean ± SEM, n = 3 independent experiments for each condition).


( B ) of of PM signals to signals of wild - type EGFP - GRAM1b ( WT ) mutant versions of EGFP - GRAM1b ( R166L , Q173I , F182I , T184V , F186L , G187L , A188S ) , as assessed by confocal microscopy scan from GRAMD1 TKO HeLa , expressing indicated constructs , as shown in ( C ) Fig EV4A ( mean ± , n = 10 for each condition ; data are pooled from one experiment ; Dunnett ' s multiple comparisons , * * P < 0 . 0001 ) .

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

(B) Quantification of the ratio of PM signals to the cytosolic signals of wild-type EGFP-GRAM1b (WT) and mutant versions of EGFP-GRAM1b (R166L, Q173I, F182I, T184V, F186L, G187L, A188S), as assessed by confocal microscopy and line scan analysis from GRAMD1 TKO HeLa cells, expressing indicated constructs, as shown in (C) and Fig EV4A (mean ± SEM, n = 10 cells for each condition; data are pooled from one experiment; Dunnett's multiple comparisons test, **P < 0.0001).


( D ) Confocal of live wild - type ( control ) GRAMD1 TKO HeLa , expressing either wild - type EGFP - GRAM1b ( WT ) or mutant EGFP - GRAM1b ( G187L ) , with or without SMase treatment ( 100 mU / ml for 1 hour at 37 ° C ) . Insets show at higher magnification regions indicated by dashed boxes . Note recruitment of mutant EGFP - GRAM1b ( G187L ) to PM even in wild - type HeLa at , which was further enhanced by SMase treatment . Scale bars , 10 µm .

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 B-MEASURED_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 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

(D) Confocal images of live wild-type (control) and GRAMD1 TKO HeLa cells, expressing either wild-type EGFP-GRAM1b (WT) or mutant EGFP-GRAM1b (G187L), with or without SMase treatment (100 mU/ml for 1 hour at 37°C). Insets show at higher magnification the regions indicated by white dashed boxes. Note the recruitment of mutant EGFP-GRAM1b (G187L) to the PM even in wild-type HeLa cells at rest, which was further enhanced by SMase treatment. Scale bars, 10 µm.


( E ) of of PM signals to signals of wild - type EGFP - GRAM1b ( WT ) mutant EGFP - GRAM1b ( G187L ) , as assessed by confocal microscopy scan from GRAMD1 TKO HeLa , expressing indicated constructs , with or without SMase treatment ( 100 mU / ml for 1 hour at 37 ° C ) , as shown in ( D ) ( mean ± , n = 10 for each condition ; data are pooled from one experiment ) .

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

(E) Quantification of the ratio of PM signals to the cytosolic signals of wild-type EGFP-GRAM1b (WT) and mutant EGFP-GRAM1b (G187L), as assessed by confocal microscopy and line scan analysis from GRAMD1 TKO HeLa cells, expressing indicated constructs, with or without SMase treatment (100 mU/ml for 1 hour at 37°C), as shown in (D) (mean ± SEM, n = 10 cells for each condition; data are pooled from one experiment).


( F ) A confocal image of live GRAMD1 TKO HeLa expressing mutant EGFP - GRAM1b ( G187L ) . were cultured in medium supplemented with 10 % lipoprotein deficient serum ( LPDS ) mevastatin ( 50 µM ) for 16 hours to deplete accessible cholesterol before imaging . An inset shows at higher magnification region indicated by a dashed box . Note absence of PM recruitment ( compare to ( D ) ) . Scale bars , 10 µm .

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

(F) A confocal image of live GRAMD1 TKO HeLa cells expressing mutant EGFP-GRAM1b (G187L). Cells were cultured in the medium supplemented with 10% lipoprotein deficient serum (LPDS) and mevastatin (50 µM) for 16 hours to deplete accessible cholesterol before imaging. An inset shows at higher magnification the region indicated by a white dashed box. Note the absence of PM recruitment (compare to (D)). Scale bars, 10 µm.