Figure 1Growth curve of NIH - 3T3 cells treated with TGFβ ( 2 ng / mL for all experiments ) or vehicle alone ( mock ) for the indicated time . The cell number at the indicated days relative to the number at the start of the treatment ( d0 ) is shown . The dotted line represents the cell number at d0 . NIH - 3T3 cells were treated with TGFβ or mock for 48h and incubated with O - propargyl - puromycin ( OPP ) for the last 60 min . The translation rate was determined by flow cytometry for OPP incorporation into proteins . A representative plot is shown on the left , and the quantification is shown on the right . Values are relative to mock - treated cells . Western blot of NIH - 3T3 cells treated with TGFβ or mock for 48h . Western blot of ECM from NIH - 3T3 cells grown in the presence of absence of TGFβ and of cell lysates from NIH - 3T3 cells grown on a parallel plate . Note the absence of tubulin and the electromobility shift of collagens in ECM extracts . ECM was produced from NIH - 3T3 cells according to ( d ) , and collagen abundance was measured by picrosirius red staining , normalized to the packed cell volume before ECM extraction , and expressed relative to mock - treated cells . NIH - 3T3 cells were treated with TGFβ or mock for 48h . The medium was replaced in the last 12h of the treatment ( with the same treatments as before ) , and glucose consumption from the fresh media were measured using the YSI bioanalyzer . ATP production rate from glycolysis ( JATPgly ) and mitochondrial oxidative phosphorylation ( JATPox ) from NIH - 3T3 cells treated with TGFβ or mock for 48h , calculated using Seahorse data shown in ( k ) and EV Figure 1f . Pearson correlation of mitochondrial ATP production rate ( JATPox ) and OPP incorporation of NIH - 3T3 cells treated with TGFβ or mock for 2h , 4h , 8h , 24h and 48h , relative to mock - treated cells . Also see EV Figure 1k and l . Note that the datapoints of the 2h and 4h measurements overlap . NIH - 3T3 cells were treated with TGFβ or mock for 48h . The medium was replaced in the last 12h of the treatment ( with the same treatments as before ) Glutamine consumption measured as in ( g ) . NIH - 3T3 cells were treated with TGFβ or mock for 48h , and the oxygen consumption rate ( OCR ) before and after treatment with mitochondrial inhibitors was measured using the Seahorse bioanalyzer . Oligo , Oligomycin ; Rot / Anti - A , Rotenone / Antimycin - A ; PCV , packed cell volume . 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 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 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1Growth curve of NIH-3T3 cells treated with TGFβ (2 ng/mL for all experiments) or vehicle alone (mock) for the indicated time. The cell number at the indicated days relative to the number at the start of the treatment (d0) is shown. The dotted line represents the cell number at d0.NIH-3T3 cells were treated with TGFβ or mock for 48h and incubated with O-propargyl-puromycin (OPP) for the last 60 min. The translation rate was determined by flow cytometry for OPP incorporation into proteins. A representative plot is shown on the left, and the quantification is shown on the right. Values are relative to mock-treated cells.Western blot of NIH-3T3 cells treated with TGFβ or mock for 48h.Western blot of ECM from NIH-3T3 cells grown in the presence of absence of TGFβ and of cell lysates from NIH-3T3 cells grown on a parallel plate. Note the absence of tubulin and the electromobility shift of collagens in ECM extracts.ECM was produced from NIH-3T3 cells according to (d), and collagen abundance was measured by picrosirius red staining, normalized to the packed cell volume before ECM extraction, and expressed relative to mock-treated cells.NIH-3T3 cells were treated with TGFβ or mock for 48h. The medium was replaced in the last 12h of the treatment (with the same treatments as before), and glucose consumption from the fresh media were measured using the YSI bioanalyzer.ATP production rate from glycolysis (JATPgly) and mitochondrial oxidative phosphorylation (JATPox) from NIH-3T3 cells treated with TGFβ or mock for 48h, calculated using Seahorse data shown in (k) and EV Figure 1f.Pearson correlation of mitochondrial ATP production rate (JATPox) and OPP incorporation of NIH-3T3 cells treated with TGFβ or mock for 2h, 4h, 8h, 24h and 48h, relative to mock-treated cells. Also see EV Figure 1k and l. Note that the datapoints of the 2h and 4h measurements overlap.NIH-3T3 cells were treated with TGFβ or mock for 48h. The medium was replaced in the last 12h of the treatment (with the same treatments as before) Glutamine consumption measured as in (g).NIH-3T3 cells were treated with TGFβ or mock for 48h, and the oxygen consumption rate (OCR) before and after treatment with mitochondrial inhibitors was measured using the Seahorse bioanalyzer. Oligo, Oligomycin; Rot/Anti-A, Rotenone/Antimycin-A; PCV, packed cell volume.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 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.


Figure 2Comparison 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 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 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 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 cells treated with TGFβ or mock for 48h , relative to mock - treated cells . Metabolites were measured by GC - MS . 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 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 .

O B-PANEL_START O O O 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-PANEL_START O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O 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-PANEL_START O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2Comparison 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 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 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 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 cells treated with TGFβ or mock for 48h, relative to mock-treated cells. Metabolites were measured by GC-MS.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 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.


Figure 3Western 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 cells expressing sgCtrl , sgP5CS - 1 or sgP5CS - 2 were treated with TGFβ or mock for 48h , and proline abundance was measured by GC - MS . Values are relative to mock - treated sgCtrl expressing cells . Right : western blot of NIH - 3T3 cells expressing sgCtrl , sgP5CS - 1 or sgP5CS - 2 . Western blot of NIH - 3T3 cells expressing sgCtrl or sgP5CS - 2 and treated with TGFβ or mock for 48h in the presence or absence of 0 . 15 mM proline . Collagen abundance in ECM produced by NIH - 3T3 cells expressing sgCtrl or sgP5CS - 2 grown in the presence or absence of TGFβ and 0 . 15 mM proline , measured by picrosirius red staining and normalized to the packed cell volume of cells grown on a parallel plate under identical conditions . Values are relative to mock - treated sgCtrl expressing cells . Proline abundance in NIH - 3T3 cells expressing empty vector or HA - P5CS cDNA , measured by GC - MS . Values are relative to mock - treated empty vector expressing cells . Western blot of NIH - 3T3 cells expressing empty vector or HA - P5CS cDNA . Collagen abundance in ECM produced by NIH - 3T3 cells expressing empty vector or HA - P5CS cDNA , measured by picrosirius red staining and normalized to the packed cell volume of cells grown on a parallel plate under identical conditions . Values are relative to mock - treatedAnalysis of the indicated gene expression datasets for mRNA levels of P5CS : ( i ) lung tissue from mice with pulmonary fibrosis induced by bleomycin ( Bleo ) treatment compared to saline treatment ( GSE112827 ) ; two datasets ( GSE110147 , GSE32537 ) from lungs of patients with idiopathic pulmonary fibrosis ( IPF ) compared to normal controls ( Ctrl ) . AU , arbitrary units . The number of patients per group is indicated . Pearson correlation of P5CS mRNA level and forced vital capacity ( FVC ) before bronchodilator ( pre - BD ) as percentage of what was predicted for each patient , from clinical data of GSE32537 . Tracing of [ U - 13C ] L - glutamine ( [ U - 13C ] Gln ) into indicated metabolites . NIH - 3T3 cells expressing sgCtrl , sgSmad4 - 1 or sgSmad4 - 2 were treated with TGFβ or mock for 48h and the medium was replaced ( including all treatments ) with DMEM without L - glutamine and supplemented with [ U - 13C ] Gln for the last 8h . Metabolites were measured by GC - MS .

O B-PANEL_START O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3Western 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 cells expressing sgCtrl, sgP5CS-1 or sgP5CS-2 were treated with TGFβ or mock for 48h, and proline abundance was measured by GC-MS. Values are relative to mock-treated sgCtrl expressing cells. Right: western blot of NIH-3T3 cells expressing sgCtrl, sgP5CS-1 or sgP5CS-2.Western blot of NIH-3T3 cells expressing sgCtrl or sgP5CS-2 and treated with TGFβ or mock for 48h in the presence or absence of 0.15 mM proline.Collagen abundance in ECM produced by NIH-3T3 cells expressing sgCtrl or sgP5CS-2 grown in the presence or absence of TGFβ and 0.15 mM proline, measured by picrosirius red staining and normalized to the packed cell volume of cells grown on a parallel plate under identical conditions. Values are relative to mock-treated sgCtrl expressing cells.Proline abundance in NIH-3T3 cells expressing empty vector or HA-P5CS cDNA, measured by GC-MS. Values are relative to mock-treated empty vector expressing cells.Western blot of NIH-3T3 cells expressing empty vector or HA-P5CS cDNA.Collagen abundance in ECM produced by NIH-3T3 cells expressing empty vector or HA-P5CS cDNA, measured by picrosirius red staining and normalized to the packed cell volume of cells grown on a parallel plate under identical conditions. Values are relative to mock-treatedAnalysis of the indicated gene expression datasets for mRNA levels of P5CS: (i) lung tissue from mice with pulmonary fibrosis induced by bleomycin (Bleo) treatment compared to saline treatment (GSE112827);two datasets (GSE110147, GSE32537) from lungs of patients with idiopathic pulmonary fibrosis (IPF) compared to normal controls (Ctrl). AU, arbitrary units. The number of patients per group is indicated.Pearson correlation of P5CS mRNA level and forced vital capacity (FVC) before bronchodilator (pre-BD) as percentage of what was predicted for each patient, from clinical data of GSE32537.Tracing of [U-13C] L-glutamine ([U-13C] Gln) into indicated metabolites. NIH-3T3 cells expressing sgCtrl, sgSmad4-1 or sgSmad4-2 were treated with TGFβ or mock for 48h and the medium was replaced (including all treatments) with DMEM without L-glutamine and supplemented with [U-13C] Gln for the last 8h. Metabolites were measured by GC-MS.


Figure 4Mitochondrial basal respiration ( a ) and FCCP - induced maximal respiration ( b ) of NIH - 3T3 cells treated with TGFβ ( 2 ng / mL for all experiments ) or mock for 2h , 4h , 8h , 24h or 48h , based on Seahorse OCR measurements . The relative values were calculated by normalizing the respective values of TGFβ - treated cells to those of mock - treated cells for each time point . Left : western blot of NIH - 3T3 cells expressing empty vector , mitoTPNOX or mitoLbNOX . MitoTPNOX or mitoLbNOX expression is detected by probing for their FLAG - tag . Right : NIH - 3T3 cells expressing empty vector , mitoTPNOX or mitoLbNOX were treated with TGFβ or mock for 48h and proline abundance was measured by GC - MS . Values are normalized to mock - treated empty vector expressing cells . NIH - 3T3 cells were treated with rotenone , antimycin , myxothiazol or vehicle control for 1h , and proline abundance was measured by GC - MS . Values are normalized to control - treated cells . NIH - 3T3 cells were treated with cobalt chloride ( CoCl2 ) or vehicle control for 6h , and proline abundance was measured by GC - MS . Values are normalized to control - treated cells . NIH - 3T3 cells were treated with TGFβ or mock for 48h , and FCCP or vehicle control was added for the last 6h of the treatment . Proline abundance was measured by GC - MS . Values are normalized to mock - and control - treated cells . NIH - 3T3 cells were treated with TGFβ or mock for 48h , and oligomycin or vehicle control was added for the last 1h or 6h of the treatment . Proline abundance was measured by GC - MS . Values are normalized to mock - and control - treated cells .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4Mitochondrial basal respiration (a) and FCCP-induced maximal respiration (b) of NIH-3T3 cells treated with TGFβ (2 ng/mL for all experiments) or mock for 2h, 4h, 8h, 24h or 48h, based on Seahorse OCR measurements. The relative values were calculated by normalizing the respective values of TGFβ-treated cells to those of mock-treated cells for each time point.Left: western blot of NIH-3T3 cells expressing empty vector, mitoTPNOX or mitoLbNOX. MitoTPNOX or mitoLbNOX expression is detected by probing for their FLAG-tag. Right: NIH-3T3 cells expressing empty vector, mitoTPNOX or mitoLbNOX were treated with TGFβ or mock for 48h and proline abundance was measured by GC-MS. Values are normalized to mock-treated empty vector expressing cells.NIH-3T3 cells were treated with rotenone, antimycin, myxothiazol or vehicle control for 1h, and proline abundance was measured by GC-MS. Values are normalized to control-treated cells.NIH-3T3 cells were treated with cobalt chloride (CoCl2) or vehicle control for 6h, and proline abundance was measured by GC-MS. Values are normalized to control-treated cells.NIH-3T3 cells were treated with TGFβ or mock for 48h, and FCCP or vehicle control was added for the last 6h of the treatment. Proline abundance was measured by GC-MS. Values are normalized to mock- and control-treated cells.NIH-3T3 cells were treated with TGFβ or mock for 48h, and oligomycin or vehicle control was added for the last 1h or 6h of the treatment. Proline abundance was measured by GC-MS. Values are normalized to mock- and control-treated cells.


Figure 5NIH - 3T3 cells were treated with TGFβ or mock for 1h , 6h , 24h or 48h . ROS levels were measured by flow cytometry for CM - H2DCFDA after incubation with CM - H2DCFDA for the last 30 min of the treatment ( Red line ) . Proline abundance was measured by GC - MS ( Blue line ) . The relative levels were calculated by normalizing the respective values of TGFβ - treated cells to those of mock - treated cells for each time point . Western blot of NIH - 3T3 cells were treated with TGFβ or mock for 1h , 6h , 24h or 48h . NIH - 3T3 cells expressing sgCtrl , sgSmad4 - 1 or sgSmad4 - 2 were treated with TGFβ or mock for 6h , and ROS levels were measured by flow cytometry for CM - H2DCFDA after incubation with CM - H2DCFDA for the last 30 min of the treatment . Values are normalized to mock - treated sgCtrl expressing cells . Western blot of NIH - 3T3 cells expressing sgCtrl , sgSmad4 - 1 or sgSmad4 - 2 and treated with TGFβ or mock for 6h in the presence of 0 . 5 % FBS . NIH - 3T3 cells expressing mito - Grx1 - roGFP2 or cyto - Grx1 - roGFP2 were treated with TGFβ or mock for 6h or with 100 µM H2O2 as a control . Emission was measured with a 520 / 10nm filter after excitation with 405nm and 488nm lasers using flow cytometry . Oxidized roGFP2 gains excitability at 405nm and loses excitability at 488nm . Oxidation status is expressed as percentage of maximal oxidation which was determined by treating cells with 5 mM H2O2 for 5 min before harvest . NIH - 3T3 cells expressing sgCtrl or sgP5CS - 2 were treated with TGFβ or mock for 48h in the presence of 0 . 15 mM proline . ROS levels were measured by flow cytometry for CM - H2DCFDA after incubation with CM - H2DCFDA for the last 30 min of the treatment .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5NIH-3T3 cells were treated with TGFβ or mock for 1h, 6h, 24h or 48h. ROS levels were measured by flow cytometry for CM-H2DCFDA after incubation with CM-H2DCFDA for the last 30 min of the treatment (Red line). Proline abundance was measured by GC-MS (Blue line). The relative levels were calculated by normalizing the respective values of TGFβ-treated cells to those of mock-treated cells for each time point.Western blot of NIH-3T3 cells were treated with TGFβ or mock for 1h, 6h, 24h or 48h.NIH-3T3 cells expressing sgCtrl, sgSmad4-1 or sgSmad4-2 were treated with TGFβ or mock for 6h, and ROS levels were measured by flow cytometry for CM-H2DCFDA after incubation with CM-H2DCFDA for the last 30 min of the treatment. Values are normalized to mock-treated sgCtrl expressing cells.Western blot of NIH-3T3 cells expressing sgCtrl, sgSmad4-1 or sgSmad4-2 and treated with TGFβ or mock for 6h in the presence of 0.5% FBS.NIH-3T3 cells expressing mito-Grx1-roGFP2 or cyto-Grx1-roGFP2 were treated with TGFβ or mock for 6h or with 100 µM H2O2 as a control. Emission was measured with a 520/10nm filter after excitation with 405nm and 488nm lasers using flow cytometry. Oxidized roGFP2 gains excitability at 405nm and loses excitability at 488nm. Oxidation status is expressed as percentage of maximal oxidation which was determined by treating cells with 5 mM H2O2 for 5 min before harvest.NIH-3T3 cells expressing sgCtrl or sgP5CS-2 were treated with TGFβ or mock for 48h in the presence of 0.15 mM proline. ROS levels were measured by flow cytometry for CM-H2DCFDA after incubation with CM-H2DCFDA for the last 30 min of the treatment.


Figure 6NIH - 3T3 cells were treated with vehicle control or CTPi at increasing concentrations ( 0 . 1 mM , 0 . 2 mM , 0 . 5 mM ) or TGFβ ( 2 ng / mL for all experiments ) for 6h , and ROS levels were measured by flow cytometry for CM - H2DCFDA after incubation with CM - H2DCFDA for the last 30 min of the treatment . Values are relative to control - treated cells . P - values represent comparison of individual samples to the control . Left : NIH - 3T3 cells expressing sgCtrl , sgSlc25a1 - 1 or sgSlc25a1 - 2 were treated with TGFβ or mock for 6h , and ROS levels were measured by flow cytometry for CM - H2DCFDA after incubation with CM - H2DCFDA for the last 30 min of the treatment . Values are relative to mock - treated sgCtrl expressing cells . Right : western blot of NIH - 3T3 cells expressing sgCtrl , sgSlc25a1 - 1 or sgSlc25a1 - 2 . NIH - 3T3 cells expressing sgCtrl , sgSlc25a1 - 1 or sgSlc25a1 - 2 were treated with TGFβ or mock for 48h and proline abundance was measured by GC - MS . Values are relative to mock - treated sgCtrl expressing cells . Western blot of NIH - 3T3 cells expressing sgCtrl , sgSlc25a1 - 1 or sgSlc25a1 - 2 and treated with TGFβ or mock for 48h . Collagen abundance in ECM produced by NIH - 3T3 cells expressing sgCtrl , sgSlc25a1 - 1 or sgSlc25a1 - 2 grown in the presence or absence of TGFβ , measured by picrosirius red staining and normalized to the packed cell volume of cells grown on a parallel plate under identical conditions . Values are relative to mock - treated sgCtrl expressing cells . NIH - 3T3 cells expressing empty vector or Slc25a1 cDNA were treated with TGFβ or mock for 6h , and ROS levels were measured by flow cytometry for CM - H2DCFDA after incubation with CM - H2DCFDA for the last 30 min of the treatment . Values are relative to mock - treated empty vector expressing cells . NIH - 3T3 cells expressing empty vector or Slc25a1 cDNA were treated with TGFβ or mock for 48h , and proline abundance was measured by GC - MS . Values are relative to mock - treated empty vector expressing cells . Western blot of NIH - 3T3 cells expressing empty vector or Slc25a1 cDNA and treated with TGFβ or mock for 48h . Collagen abundance in ECM produced by NIH - 3T3 cells expressing empty vector or Slc25a1 cDNA grown in the presence or absence of TGFβ , measured by picrosirius red staining and normalized to the packed cell volume of cells grown on a parallel plate under identical conditions . Values are relative to mock - treated empty vector expressing cells . Analysis of the indicated gene expression datasets for mRNA levels of Slc25a1 , as described in Figure 3 : ( j ) lung tissue from mice with pulmonary fibrosis induced by bleomycin ( Bleo ) treatment compared to saline treatment ( GSE112827 ) ; Analysis of the indicated gene expression datasets for mRNA levels of Slc25a1 , as described in Figure 3 : two datasets ( GSE110147 , GSE32537 ) from lungs of patients with idiopathic pulmonary fibrosis ( IPF ) compared to normal controls ( Ctrl ) . AU , arbitrary units . The number of patients per group is indicated . Pearson correlation of Slc25a1 and P5CS mRNA levels in IPF patients from GSE32537 . IPF patients from GSE32537 were assigned into P5CSlow / SLC25A1high and P5CShigh / SLC25A1low groups based on the expression level of P5CS and SLC25A1 . " High " represents patients with P5CS or SLC25A1 expression values being above the 75 % percentile of the respective gene expression ; " low " represents patients with expression values being below the 25 % percentile of gene expression . The forced vital capacity ( FVC ) before bronchodilator ( pre - BD ) as percentage of what was predicted for each patient was compared between the groups .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O 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-PANEL_START O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6NIH-3T3 cells were treated with vehicle control or CTPi at increasing concentrations (0.1 mM, 0.2 mM, 0.5 mM) or TGFβ (2 ng/mL for all experiments) for 6h, and ROS levels were measured by flow cytometry for CM-H2DCFDA after incubation with CM-H2DCFDA for the last 30 min of the treatment. Values are relative to control-treated cells. P-values represent comparison of individual samples to the control.Left: NIH-3T3 cells expressing sgCtrl, sgSlc25a1-1 or sgSlc25a1-2 were treated with TGFβ or mock for 6h, and ROS levels were measured by flow cytometry for CM-H2DCFDA after incubation with CM-H2DCFDA for the last 30 min of the treatment. Values are relative to mock-treated sgCtrl expressing cells. Right: western blot of NIH-3T3 cells expressing sgCtrl, sgSlc25a1-1 or sgSlc25a1-2.NIH-3T3 cells expressing sgCtrl, sgSlc25a1-1 or sgSlc25a1-2 were treated with TGFβ or mock for 48h and proline abundance was measured by GC-MS. Values are relative to mock-treated sgCtrl expressing cells.Western blot of NIH-3T3 cells expressing sgCtrl, sgSlc25a1-1 or sgSlc25a1-2 and treated with TGFβ or mock for 48h.Collagen abundance in ECM produced by NIH-3T3 cells expressing sgCtrl, sgSlc25a1-1 or sgSlc25a1-2 grown in the presence or absence of TGFβ, measured by picrosirius red staining and normalized to the packed cell volume of cells grown on a parallel plate under identical conditions. Values are relative to mock-treated sgCtrl expressing cells.NIH-3T3 cells expressing empty vector or Slc25a1 cDNA were treated with TGFβ or mock for 6h, and ROS levels were measured by flow cytometry for CM-H2DCFDA after incubation with CM-H2DCFDA for the last 30 min of the treatment. Values are relative to mock-treated empty vector expressing cells.NIH-3T3 cells expressing empty vector or Slc25a1 cDNA were treated with TGFβ or mock for 48h, and proline abundance was measured by GC-MS. Values are relative to mock-treated empty vector expressing cells.Western blot of NIH-3T3 cells expressing empty vector or Slc25a1 cDNA and treated with TGFβ or mock for 48h.Collagen abundance in ECM produced by NIH-3T3 cells expressing empty vector or Slc25a1 cDNA grown in the presence or absence of TGFβ, measured by picrosirius red staining and normalized to the packed cell volume of cells grown on a parallel plate under identical conditions. Values are relative to mock-treated empty vector expressing cells.Analysis of the indicated gene expression datasets for mRNA levels of Slc25a1, as described in Figure 3: (j) lung tissue from mice with pulmonary fibrosis induced by bleomycin (Bleo) treatment compared to saline treatment (GSE112827);Analysis of the indicated gene expression datasets for mRNA levels of Slc25a1, as described in Figure 3: two datasets (GSE110147, GSE32537) from lungs of patients with idiopathic pulmonary fibrosis (IPF) compared to normal controls (Ctrl). AU, arbitrary units. The number of patients per group is indicated.Pearson correlation of Slc25a1 and P5CS mRNA levels in IPF patients from GSE32537. IPF patients from GSE32537 were assigned into P5CSlow/SLC25A1high and P5CShigh/SLC25A1low groups based on the expression level of P5CS and SLC25A1. "High" represents patients with P5CS or SLC25A1 expression values being above the 75% percentile of the respective gene expression; "low" represents patients with expression values being below the 25% percentile of gene expression. The forced vital capacity (FVC) before bronchodilator (pre-BD) as percentage of what was predicted for each patient was compared between the groups.


Figure 1 ( 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 ) 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 ) 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 ) .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1(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) 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) 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).


Figure 2 ( 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 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 .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2(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 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.


Figure 3Effects 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 ● .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3Effects 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 ●.


Figure 4 ( 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 ) 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 .

O O B-PANEL_START O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4(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) 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.


Figure 5 ( 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 ) 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 .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5(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) 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.


Figure 1Coronal 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 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 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 ) 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 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 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 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 ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1Coronal 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 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 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) 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 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 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 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).


Figure 2Quantification 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 ) . 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 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 + cells in the VZ ( yellow circle ) and forming ectopic rosettes ( yellow box ) . 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 the nodule , which is surrounded by thickened GFP + processes ( pink arrows ) , Arl13b staining shows the formation of an ectopic apical surfaceReduction 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 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 ) . 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 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O 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-PANEL_START O 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-PANEL_START O O O O O O O O O O O O O O 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-PANEL_START O O O O 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-PANEL_START O 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-PANEL_START O O O O O 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-PANEL_START O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2Quantification 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).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 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+ cells in the VZ (yellow circle) and forming ectopic rosettes (yellow box).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 the nodule, which is surrounded by thickened GFP+ processes (pink arrows), Arl13b staining shows the formation of an ectopic apical surfaceReduction 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 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).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.


Figure 3Brightfield images of 60d old CTRL and ECE2 KO COs show normal formation of neuroepithelial structures . ISH for ECE2 confirms the absence of ECE2 mRNA in 60d old ECE2 KO COs ( pink dotted line indicates position of the CP ' ) . , 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 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 + 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 ) .

O B-PANEL_START O O O O O O O O O O O O O O O O B-PANEL_START O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3Brightfield images of 60d old CTRL and ECE2 KO COs show normal formation of neuroepithelial structures.ISH for ECE2 confirms the absence of ECE2 mRNA in 60d old ECE2 KO COs (pink dotted line indicates position of the CP')., 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 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+ 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).


Figure 4Quantification 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 - 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 ) . 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 ) . 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 ) . 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 ) . 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 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 ) . 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 ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4Quantification 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- 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).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).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).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).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 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).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).


Figure 5Binning 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 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 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 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 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 ' .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5Binning 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 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 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 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 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'.


Figure 6Volcano 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 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 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6Volcano 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 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.


Figure 1 ( 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 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 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 ] .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1(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 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 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].


Figure 2 ( 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 .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2(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.


Figure 3 ( 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 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 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 ) 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 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 ] .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3(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 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 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) 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 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].


Figure 4 ( 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 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 /

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4(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 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