( A ) Confocal imaging showed anti - GA antibody treatment reduces poly - GA aggregates and TDP - 43 mislocalization in hippocampal neurons . White and red arrows show cells with cytoplasmic TDP - 43 in GFP - positive and - negative cells , respectively . Scale bar denotes 30 µm .

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(A) Confocal imaging showed anti-GA antibody treatment reduces poly-GA aggregates and TDP-43 mislocalization in hippocampal neurons. White and red arrows show cells with cytoplasmic TDP-43 in GFP-positive and -negative cells, respectively. Scale bar denotes 30 µm.


( B ) Automated quantification of cells with cytoplasmic TDP - 43 in GFP or GA175 - GFP transduced ( donor ) , non - transduced ( receiver ) neurons . Four groups were excluded due to very high GFP transduction rate ( GFP - negative donor ) and very low GFP transmission rate ( GFP - positive receiver with IgG and anti - GA ) and complete prevention of GA - RFP transmission of anti - GA immunodepletion ( GA - GFP receiver with anti - GA ) . n = 3 biological replicates . In total 280 donor GFP , 284 receiver GFP with IgG , 317 receiver GFP with anti - GA , 277 donor GA175 - GFP , 294 receiver GA175 - GFP with IgG , 311 receiver GA175 - GFP with anti - GA cells were analyzed . Scatter plot with bar - graphs of mean ± SD . One - way ANOVA with Tukey ' s multiple comparisons test . * * * denotes p < 0 . 001 .

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(B) Automated quantification of cells with cytoplasmic TDP-43 in GFP or GA175-GFP transduced (donor), non-transduced (receiver) neurons. Four groups were excluded due to very high GFP transduction rate (GFP-negative donor) and very low GFP transmission rate (GFP-positive receiver with IgG and anti-GA) and complete prevention of GA-RFP transmission of anti-GA immunodepletion (GA-GFP receiver with anti-GA). n=3 biological replicates. In total 280 donor GFP, 284 receiver GFP with IgG, 317 receiver GFP with anti-GA, 277 donor GA175-GFP, 294 receiver GA175-GFP with IgG, 311 receiver GA175-GFP with anti-GA cells were analyzed. Scatter plot with bar-graphs of mean ± SD. One-way ANOVA with Tukey's multiple comparisons test. *** denotes p<0.001.


( C ) Immunoblotting of poly - GA immunoprecipitated from conditioned media using antibody - coupled beads and antibody leftover pre and post immunoprecipitation in the conditioned media .

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(C) Immunoblotting of poly-GA immunoprecipitated from conditioned media using antibody-coupled beads and antibody leftover pre and post immunoprecipitation in the conditioned media.


( D ) Poly - GA levels in conditioned media before and after immunodepletion with control IgG and anti - GA antibody were determined by immunoassay . n = 3 biological replicates . Scatter plot with bar - graphs of mean ± SD . One - way ANOVA with Tukey ' s multiple comparisons test . * * denotes p < 0 . 01 , and * * * denotes p < 0 . 001 .

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(D) Poly-GA levels in conditioned media before and after immunodepletion with control IgG and anti-GA antibody were determined by immunoassay. n=3 biological replicates. Scatter plot with bar-graphs of mean ± SD. One-way ANOVA with Tukey's multiple comparisons test. ** denotes p<0.01, and *** denotes p<0.001.


Double immunofluorescence of the proteasome subunit PSMC4 and poly - GA inclusions in spinal cord of GA149 - CFP transgenic mouse Scale bar denotes 20 µm .

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Double immunofluorescence of the proteasome subunit PSMC4 and poly-GA inclusions in spinal cord of GA149-CFP transgenic mouse Scale bar denotes 20 µm.


Double immunofluorescence of the proteasome subunit PSMC4 and poly - GA inclusions in cortex of C9orf72 patients compared to controls . Scale bar denotes 20 µm .

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Double immunofluorescence of the proteasome subunit PSMC4 and poly-GA inclusions in cortex of C9orf72 patients compared to controls. Scale bar denotes 20 µm.


Co - culture model of HeLa cells transfected with iRFP or GA175 - iRFP in the donor compartment and an UbG76V - GFP proteostasis reporter in donor and receiver compartment ( 48 h ) . ( C ) Separate analysis of both compartments by immunoblot and ( D ) immunoblot quantification . For quantitative analysis of immunoblots UbG76V - GFP was normalized to calnexin . n = 3 biological replicates . Scatter plot with mean ± SD . One - way ANOVA with Tukey ' s multiple comparisons test . Red dashed line indicates the control ' s expression level . * denotes p < 0 . 05 , * * * denotes p < 0 . 001 .

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Co-culture model of HeLa cells transfected with iRFP or GA175-iRFP in the donor compartment and an UbG76V-GFP proteostasis reporter in donor and receiver compartment (48 h). (C) Separate analysis of both compartments by immunoblot and (D) immunoblot quantification. For quantitative analysis of immunoblots UbG76V-GFP was normalized to calnexin. n=3 biological replicates. Scatter plot with mean ± SD. One-way ANOVA with Tukey's multiple comparisons test. Red dashed line indicates the control's expression level. * denotes p<0.05, *** denotes p<0.001.


( E ) GFP mRNA expression levels were measured by qPCR . RNA levels were normalized to GAPDH , β - actin and β2 - microglobulin mRNA . Bar - graphs of mean ± SD . n = 3 biological replicates . Unpaired two - tailed t - test with Welch ' s correction .

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(E) GFP mRNA expression levels were measured by qPCR. RNA levels were normalized to GAPDH, β-actin and β2-microglobulin mRNA. Bar-graphs of mean ± SD. n=3 biological replicates. Unpaired two-tailed t-test with Welch's correction.


( F , G ) Flow cytometry analysis of a UbG76V - GFP reporter cell line incubated 48h with conditioned media from RFP or GA175 - RFP transfected cells upon immunodepletion of poly - GA or control depletion using unspecific IgG . n = 3 biological replicates . Scatter plot with bar - graphs of mean ± SD . One - way ANOVA with Tukey ' s multiple comparisons test . * denotes p < 0 . 05 . ( G ) Comparisons of the corresponding histograms for compensated RFP and UbG76V - GFP fluorescence from one representative experiment that shows specific transmission of GA175 - RFP associated with accumulation of UbG76V - GFP in cells incubated with GA175 - RFP conditioned media .

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(F,G) Flow cytometry analysis of a UbG76V-GFP reporter cell line incubated 48h with conditioned media from RFP or GA175-RFP transfected cells upon immunodepletion of poly-GA or control depletion using unspecific IgG. n=3 biological replicates. Scatter plot with bar-graphs of mean ± SD. One-way ANOVA with Tukey's multiple comparisons test. * denotes p<0.05. (G) Comparisons of the corresponding histograms for compensated RFP and UbG76V-GFP fluorescence from one representative experiment that shows specific transmission of GA175-RFP associated with accumulation of UbG76V-GFP in cells incubated with GA175-RFP conditioned media.


Primary hippocampal neurons were transduced with GFP or GA175 - GFP after 4 days in vitro , incubated for 7 days ( DIV 4 + 7 ) , and treated with vehicle ( DMSO ) , MG132 ( 10µM ) or rolipram ( 30 µM ) for 16h . ( A ) Immunofluorescence reveals enhanced cytoplasmic TDP - 43 levels in neurons with poly - GA aggregates or treated with MG132 . Arrows mark punctate TDP - 43 staining . Rolipram treatment reduced cytoplasmic TDP - 43 in GA175 - GFP neurons . Scale bar denotes 20 µm . ( B ) Automated quantification of cells with cytoplasmic TDP - 43 in GFP or GA175 - GFP transduced neurons . n = 4 biological replicates . In total 462 GFP and 371 GA175 - GFP cells treated with vehicle , and 386 GFP and 529 GA175 - GFP cells treated with MG132 , 513 GFP and 434 GA175 - GFP cells treated with rolipram were analyzed . Scatter plot with bar - graphs of mean ± SD . One - way ANOVA with Tukey ' s multiple comparisons test .

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Primary hippocampal neurons were transduced with GFP or GA175-GFP after 4 days in vitro, incubated for 7 days (DIV 4+7), and treated with vehicle (DMSO), MG132 (10µM) or rolipram (30 µM) for 16h. (A) Immunofluorescence reveals enhanced cytoplasmic TDP-43 levels in neurons with poly-GA aggregates or treated with MG132. Arrows mark punctate TDP-43 staining. Rolipram treatment reduced cytoplasmic TDP-43 in GA175-GFP neurons. Scale bar denotes 20 µm. (B) Automated quantification of cells with cytoplasmic TDP-43 in GFP or GA175-GFP transduced neurons. n=4 biological replicates. In total 462 GFP and 371 GA175-GFP cells treated with vehicle, and 386 GFP and 529 GA175-GFP cells treated with MG132, 513 GFP and 434 GA175-GFP cells treated with rolipram were analyzed. Scatter plot with bar-graphs of mean ± SD. One-way ANOVA with Tukey's multiple comparisons test.


Primary hippocampal neurons were transduced with GFP or GA175 - GFP after 4 days in vitro , incubated for 7 days ( DIV 4 + 7 ) , and treated with vehicle ( DMSO ) , MG132 ( 10µM ) or rolipram ( 30 µM ) for 16h . ( C ) Immunoblot to show effects of MG132 and rolipram on GA175 - GFP and GFP expression .

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Primary hippocampal neurons were transduced with GFP or GA175-GFP after 4 days in vitro, incubated for 7 days (DIV 4+7), and treated with vehicle (DMSO), MG132 (10µM) or rolipram (30 µM) for 16h. (C) Immunoblot to show effects of MG132 and rolipram on GA175-GFP and GFP expression.


Primary hippocampal neurons were transduced with GFP or GA175 - GFP after 4 days in vitro , incubated for 7 days ( DIV 4 + 7 ) , and treated with vehicle ( DMSO ) , MG132 ( 10µM ) or rolipram ( 30 µM ) for 16h . Filter trap assay with quantification of SDS - insoluble aggregated GA175 - GFP . n = 5 biological replicates . Scatter plot with mean ± SD . One - way ANOVA with Tukey ' s multiple comparisons test .

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Primary hippocampal neurons were transduced with GFP or GA175-GFP after 4 days in vitro, incubated for 7 days (DIV 4+7), and treated with vehicle (DMSO), MG132 (10µM) or rolipram (30 µM) for 16h. Filter trap assay with quantification of SDS-insoluble aggregated GA175-GFP. n=5 biological replicates. Scatter plot with mean ± SD. One-way ANOVA with Tukey's multiple comparisons test.


( F , G ) HeLa cells were co - transfected with RFP - TDP - CTF and GFP or GA175 - GFP for 2 days . For the final 16h , cells were treated with rolipram ( 30 µM ) or MG132 ( 10 µM ) . Filter trap assay of SDS - insoluble TDP - CTF aggregates quantified by densitometry . n = 4 biological replicates . Scatter dot plot , mean ± SD . One - way ANOVA with Tukey ' s multiple comparisons test .

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(F,G) HeLa cells were co-transfected with RFP-TDP-CTF and GFP or GA175-GFP for 2 days. For the final 16h, cells were treated with rolipram (30 µM) or MG132 (10 µM). Filter trap assay of SDS-insoluble TDP-CTF aggregates quantified by densitometry. n=4 biological replicates. Scatter dot plot, mean ± SD. One-way ANOVA with Tukey's multiple comparisons test.


( H , I ) HeLa cells were co - transfected with TDP - 43ΔNLS - GFP and iRFP or GA175 - iRFP for 2 days . For the final 16h , cells were treated with either vehicle or rolipram ( 30 µM ) . Filter trap assay of SDS - insoluble TDP - 43ΔNLS - GFP aggregates quantified by densitometry . n = 3 biological replicates . Scatter dot plot , mean ± SD . One - way ANOVA with Tukey ' s multiple comparisons test .

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(H,I) HeLa cells were co-transfected with TDP-43ΔNLS-GFP and iRFP or GA175-iRFP for 2 days. For the final 16h, cells were treated with either vehicle or rolipram (30 µM). Filter trap assay of SDS-insoluble TDP-43ΔNLS-GFP aggregates quantified by densitometry. n=3 biological replicates. Scatter dot plot, mean ± SD. One-way ANOVA with Tukey's multiple comparisons test.


Hela cells were co - transfected with an RFP - based TDP - NLS reporter and GFP or GA175 - GFP . 24 h after transfection , cells were treated with rolipram ( 30 µM ) for 16h . In the immunofluorescence GFP is not shown because diffuse GFP expression would hide the cytoplasmic RFP - reporter . White arrows indicate cells with cytoplasmic TDP - 43 . ( B ) Automated quantification of cells with cytoplasmic TDP - NLS reporter in GFP and GA175 - GFP positive cells . n = 4 biological replicates . In total 345 GFP and 386 GA175 - GFP cells treated with vehicle , 371 GFP and 404 GA175 - GFP cells treated with rolipram were analyzed . Scatter plot with bar - graphs of mean ± SD . One - way ANOVA with Tukey ' s multiple comparisons test .

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Hela cells were co-transfected with an RFP-based TDP-NLS reporter and GFP or GA175-GFP. 24 h after transfection, cells were treated with rolipram (30 µM) for 16h. In the immunofluorescence GFP is not shown because diffuse GFP expression would hide the cytoplasmic RFP-reporter. White arrows indicate cells with cytoplasmic TDP-43. (B) Automated quantification of cells with cytoplasmic TDP-NLS reporter in GFP and GA175-GFP positive cells. n=4 biological replicates. In total 345 GFP and 386 GA175-GFP cells treated with vehicle, 371 GFP and 404 GA175-GFP cells treated with rolipram were analyzed. Scatter plot with bar-graphs of mean ± SD. One-way ANOVA with Tukey's multiple comparisons test.


Hela cells were co - transfected with an RFP - based TDP - NLS reporter and GFP or GA175 - GFP . 24 h after transfection , cells were treated with rolipram ( 30 µM ) for 16h . ( C ) RFP - NLSTDPWT mRNA expression levels were measured by qPCR . n = 3 biological replicates . Bar - graphs of mean ± SD . One - way ANOVA with Tukey ' s multiple comparisons test .

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Hela cells were co-transfected with an RFP-based TDP-NLS reporter and GFP or GA175-GFP. 24 h after transfection, cells were treated with rolipram (30 µM) for 16h. (C) RFP-NLSTDPWT mRNA expression levels were measured by qPCR. n=3 biological replicates. Bar-graphs of mean ± SD. One-way ANOVA with Tukey's multiple comparisons test.


Hela cells were co - transfected with the RFP - based TDP - NLS reporter , GFP or GA175 - GFP , as well as PSMD11 or empty vector . Image analysis as in ( A ) . n = 4 biological replicates . Scatter plot with bar - graphs of mean ± SD . One - way ANOVA with Tukey ' s multiple comparisons test . 354 GFP and 330 GA175 - GFP cells with vector , 367 GFP and 369 GA175 - GFP cells with PSMD11 in total were analyzed .

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Hela cells were co-transfected with the RFP-based TDP-NLS reporter, GFP or GA175-GFP, as well as PSMD11 or empty vector. Image analysis as in (A). n=4 biological replicates. Scatter plot with bar-graphs of mean ± SD. One-way ANOVA with Tukey's multiple comparisons test. 354 GFP and 330 GA175-GFP cells with vector, 367 GFP and 369 GA175-GFP cells with PSMD11 in total were analyzed.


Hela cells were co - transfected with the RFP - based TDP - NLS reporter , GFP or GA175 - GFP , as well as PSMD11 or empty vector . F ) RFP - NLSTDPWT mRNA expression levels were measured by qPCR . n = 3 biological replicates . Bar - graphs of mean ± SD . One - way ANOVA with Tukey ' s multiple comparisons test .

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Hela cells were co-transfected with the RFP-based TDP-NLS reporter, GFP or GA175-GFP, as well as PSMD11 or empty vector. F) RFP-NLSTDPWT mRNA expression levels were measured by qPCR. n=3 biological replicates. Bar-graphs of mean ± SD. One-way ANOVA with Tukey's multiple comparisons test.


Immunofluorescence of Hela cells transfected with GFP or GA175 - GFP showing reduced poly - GA aggregation upon rolipram treatment ( 30 µM , 16 h ) . ( H ) Automated quantification of poly - GA aggregate number per cell . n = 3 biological replicates . In total 223 cells treated with vehicle and 286 cells treated with rolipram were analyzed . Scatter plot with bar - graphs of mean ± SD . Unpaired two - tailed t - test with Welch ' s correction .

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Immunofluorescence of Hela cells transfected with GFP or GA175-GFP showing reduced poly-GA aggregation upon rolipram treatment (30 µM, 16 h). (H) Automated quantification of poly-GA aggregate number per cell. n=3 biological replicates. In total 223 cells treated with vehicle and 286 cells treated with rolipram were analyzed. Scatter plot with bar-graphs of mean ± SD. Unpaired two-tailed t-test with Welch's correction.


( I ) GA - GFP mRNA expression levels were measured by qPCR . n = 3 biological replicates . Bar - graphs of mean ± SD . Unpaired two - tailed t - test with Welch ' s correction .

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(I) GA-GFP mRNA expression levels were measured by qPCR. n=3 biological replicates. Bar-graphs of mean ± SD. Unpaired two-tailed t-test with Welch's correction.


( B ) Immunoblot of HeLa cells transfected with RFP - based TDP - NLS wild - type ( WT ) or mutants ( K84A , K84R , K95A , K95R ) .

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(B) Immunoblot of HeLa cells transfected with RFP-based TDP-NLS wild-type (WT) or mutants (K84A, K84R, K95A, K95R).


( C HeLa cells were co - transfected with the indicated TDP - NLS reporters as well as GFP or GA175 - GFP , and treated with MG132 ( 10µM ) or vehicle for 16h .

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(C HeLa cells were co-transfected with the indicated TDP-NLS reporters as well as GFP or GA175-GFP, and treated with MG132 (10µM) or vehicle for 16h.


( D ) Automated quantification of RFP - NLS reporters in GFP positive cells . Note that K84A and K84R block overall import , while K95A and K95R allow import but are resistant to inhibition by poly - GA . n = 4 biological replicates . The total number of cells analyzed per group was ( from left to right ) : 667 , 581 , 789 , 783 , 809 , 708 , 628 , 721 , 938 , 557 , 857 , 861 , 886 , 699 , 789 , 539 , 636 , 577 , 638 and 870 . Scatter plot with bar - graphs of mean ± SD . One - way ANOVA with Tukey ' s multiple comparisons test . * * * denotes p < 0 . 001 . Scale bar denotes 20 µm .

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(D) Automated quantification of RFP-NLS reporters in GFP positive cells. Note that K84A and K84R block overall import, while K95A and K95R allow import but are resistant to inhibition by poly-GA. n=4 biological replicates. The total number of cells analyzed per group was (from left to right): 667, 581, 789, 783, 809, 708, 628, 721, 938, 557, 857, 861, 886, 699,789, 539, 636, 577, 638 and 870. Scatter plot with bar-graphs of mean ± SD. One-way ANOVA with Tukey's multiple comparisons test. *** denotes p<0.001. Scale bar denotes 20 µm.


( E , F ) HeLa cells were co - transfected with the indicated GFP - TDP NLS reporters and iRFP or GA175 - iRFP . Cell lysates were immunoprecipitated with anti - GFP and immunoblotted with indicated antibodies to detect co - immunoprecipitation of the TDP - 43 NLS with importin - α5 / KPNA1 nuclear import receptor . ( F ) Quantification of KPNA1 levels normalized to total GFP - NLSTDP reporter levels in anti - GFP immunoprecipitates . n = 3 biological replicates . Scatter plot with mean ± SD . One - way ANOVA with Tukey ' s multiple comparisons test . * * * denotes p < 0 . 001 . See also Figure EV4 . Red dashed line indicates the control ' s expression level .

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(E,F) HeLa cells were co-transfected with the indicated GFP-TDP NLS reporters and iRFP or GA175-iRFP. Cell lysates were immunoprecipitated with anti-GFP and immunoblotted with indicated antibodies to detect co-immunoprecipitation of the TDP-43 NLS with importin-α5/KPNA1 nuclear import receptor. (F) Quantification of KPNA1 levels normalized to total GFP-NLSTDP reporter levels in anti-GFP immunoprecipitates. n=3 biological replicates. Scatter plot with mean ± SD. One-way ANOVA with Tukey's multiple comparisons test. *** denotes p<0.001. See also Figure EV4. Red dashed line indicates the control's expression level.


HeLa cells were co - transfected with wildtype or K95A GFP - TDP NLS , HA - ubiquitin and iRFP or GA175 - iRFP . 24h after transfection , cells were treated with rolipram ( 30 µM ) , MG132 ( 10 µM ) or DMSO ( vehicle ) for 16h . Lysates were immunoprecipitated with Protein G beads coupled with anti - GFP antibody . ( A ) Immunoblotting of input ( left panels ) and anti - GFP immunoprecipitates ( right panels ) to show GFP - reporter levels and poly - ubiquitination . ( B ) Quantification of HA - ubiquitin levels normalized to total GFP - NLSTDP reporter levels in anti - GFP immunoprecipitates . n = 3 biological replicates . Scatter plot , mean ± SD . One - way ANOVA with Tukey ' s multiple comparisons test . * denotes p < 0 . 05 , and * * * denotes p < 0 . 001 . Red dashed line indicates the control ' s expression level .

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HeLa cells were co-transfected with wildtype or K95A GFP-TDP NLS, HA-ubiquitin and iRFP or GA175-iRFP. 24h after transfection, cells were treated with rolipram (30 µM), MG132 (10 µM) or DMSO (vehicle) for 16h. Lysates were immunoprecipitated with Protein G beads coupled with anti-GFP antibody. (A) Immunoblotting of input (left panels) and anti-GFP immunoprecipitates (right panels) to show GFP-reporter levels and poly-ubiquitination. (B) Quantification of HA-ubiquitin levels normalized to total GFP- NLSTDP reporter levels in anti-GFP immunoprecipitates. n=3 biological replicates. Scatter plot, mean ± SD. One-way ANOVA with Tukey's multiple comparisons test. * denotes p<0.05, and *** denotes p<0.001. Red dashed line indicates the control's expression level.


HeLa cells were co - transfected with wildtype or K95A GFP - TDP NLS , HA - ubiquitin and iRFP or GA175 - iRFP . 24h after transfection , cells were treated with rolipram ( 30 µM ) , MG132 ( 10 µM ) or DMSO ( vehicle ) for 16h . Lysates were immunoprecipitated with Protein G beads coupled with anti - GFP antibody .

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HeLa cells were co-transfected with wildtype or K95A GFP-TDP NLS, HA-ubiquitin and iRFP or GA175-iRFP. 24h after transfection, cells were treated with rolipram (30 µM), MG132 (10 µM) or DMSO (vehicle) for 16h. Lysates were immunoprecipitated with Protein G beads coupled with anti-GFP antibody.


ABCE1IIEA efficiently splits 70S ribosomes in the presence of AMP - PNP and aRF1 / aPelota . The 30S population contains a stoichiometric ratio of ABCE1 and ribosomal proteins , forming the post - splitting complex . rps : small subunit ribosomal proteins .

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ABCE1IIEA efficiently splits 70S ribosomes in the presence of AMP-PNP and aRF1/aPelota. The 30S population contains a stoichiometric ratio of ABCE1 and ribosomal proteins, forming the post-splitting complex. rps: small subunit ribosomal proteins.


Yeast survival of ABCE1 variants ( S . solfataricus colored , S . cerevisiae in gray ) . Most residues connecting to 30S in the post - SC show a growth defect when exchanged for a small one ( alanine ) or a negative charge ( glutamate ) . + + no effect , + growth defect , - lethal .

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Yeast survival of ABCE1 variants (S. solfataricus colored, S. cerevisiae in gray). Most residues connecting to 30S in the post-SC show a growth defect when exchanged for a small one (alanine) or a negative charge (glutamate). ++ no effect, + growth defect, - lethal.


Mutations in the α - helices of hinge 2 prevent 30S binding while the Y592A / Y593A ( C - terminus ) and L353Y ( A - loop in NBSII ) exchanges do not influence ribosome binding .

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Mutations in the α-helices of hinge 2 prevent 30S binding while the Y592A/Y593A (C-terminus) and L353Y (A-loop in NBSII) exchanges do not influence ribosome binding.


70S splitting efficiency normalized to wildtype . Hinge 2 mutations Y592A / Y593A , R565E and S580E display strongly impaired splitting activity . Unspecific ribosome dissociation level as determined in control experiments in the absence of ABCE1 is marked by the dotted line . ATP turnover per ABCE1 is not affected in all tested mutants .

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70S splitting efficiency normalized to wildtype. Hinge 2 mutations Y592A/Y593A, R565E and S580E display strongly impaired splitting activity. Unspecific ribosome dissociation level as determined in control experiments in the absence of ABCE1 is marked by the dotted line. ATP turnover per ABCE1 is not affected in all tested mutants.


C . Total USV number in YFO and AFO . Data information : Data are presented in the box plot or pie chart format . The central band in each box represents the median ; boxes indicate the middle quartiles , and whiskers extend to the minimum and maximum values . * p < 0 . 05 , determined using Student ' s or Welch t - test .

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C. Total USV number in YFO and AFO. Data information: Data are presented in the box plot or pie chart format. The central band in each box represents the median; boxes indicate the middle quartiles, and whiskers extend to the minimum and maximum values. *p<0.05, determined using Student's or Welch t-test.


D . A heat map of the 16 hyper - and 96 hypo - methylated DMRs in sperm from aged mice ordered by their chromosomal positions . M indicates the average .

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D. A heat map of the 16 hyper- and 96 hypo-methylated DMRs in sperm from aged mice ordered by their chromosomal positions. M indicates the average.


A . Volcano plot for DEGs in the embryonic brain of YFO and AFO at E14 . 5 ( n = 3 in each group ) . Green lines indicate a twofold change and p = 0 . 01 . Orange dots indicate REST / NRSF target genes . Data information : Data are presented in the volcano plot or line chart format . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , determined by nominal GSEA p - value .

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A. Volcano plot for DEGs in the embryonic brain of YFO and AFO at E14.5 (n=3 in each group). Green lines indicate a twofold change and p=0.01. Orange dots indicate REST/NRSF target genes. Data information: Data are presented in the volcano plot or line chart format. *p<0.05, ** p<0.01, ***p<0.001, determined by nominal GSEA p-value.


A . Volcano plot for DEGs in the embryonic brain of YFO and AFO at E11 . 5 ( n = 3 in each group ) . Green lines indicate a twofold change and p = 0 . 01 . Orange dots indicate REST / NRSF target genes . Data information : Data are presented in the volcano plot or line chart format . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , determined by volcano or nominal GSEA P - value . NES , normalized enrichment score .

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A. Volcano plot for DEGs in the embryonic brain of YFO and AFO at E11.5 (n=3 in each group). Green lines indicate a twofold change and p=0.01. Orange dots indicate REST/NRSF target genes. Data information: Data are presented in the volcano plot or line chart format. *p<0.05, ** p<0.01, ***p<0.001, determined by volcano or nominal GSEA P-value. NES, normalized enrichment score.


C . Immunostaining for cortical layer markers in the postnatal brain of YFO and AFO ( representative magnified images of the white boxes in panel A ) . Satb2 + / Ctip2 - : layer 2 - 4 , Ctip2high : layer 5 , Satb2 - / Ctip2 - : layer 6 . Scale bar : 100 μm . F . Cell number in each cortical layer in the postnatal brain of YFO and AFO . Data information : All data are presented as the mean ± SEM . * p < 0 . 05 , * * p < 0 . 01 , determined using Student t - test .

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C. Immunostaining for cortical layer markers in the postnatal brain of YFO and AFO (representative magnified images of the white boxes in panel A). Satb2+/Ctip2-: layer 2-4, Ctip2high: layer 5, Satb2-/Ctip2-: layer 6. Scale bar: 100 μm. F. Cell number in each cortical layer in the postnatal brain of YFO and AFO. Data information: All data are presented as the mean ± SEM. *p<0.05, **p<0.01, determined using Student t-test.


D . Immunostaining for another cortical layer markers in the postnatal brain of YFO and AFO ( representative magnified images of the white boxes in panel A ) . Tbr1 - / Ctip2 - : layer 2 - 4 , Ctip2high : layer 5 , Tbr1 + / Ctip2 - : layer 6 . Scale bar : 100 μm . G . Cell density in each cortical layer in the postnatal brain of YFO and AFO . Data information : All data are presented as the mean ± SEM . * p < 0 . 05 , * * p < 0 . 01 , determined using Student t - test .

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D. Immunostaining for another cortical layer markers in the postnatal brain of YFO and AFO (representative magnified images of the white boxes in panel A). Tbr1-/Ctip2-: layer 2-4, Ctip2high: layer 5, Tbr1+/Ctip2-: layer 6. Scale bar: 100 μm. G. Cell density in each cortical layer in the postnatal brain of YFO and AFO. Data information: All data are presented as the mean ± SEM. *p<0.05, **p<0.01, determined using Student t-test.


C . Representative image of 5 - mC immunofluorescence in the sperm of saline - and 5 - Aza - treated mice ( ST and AT , respectively ) . Scale bar : 100 μm . D . Quantification analysis of 5 - mC immunofluorescence in individual sperm cells from STO ( n = 1 , 673 sperm from 4 male mice ) and ATO ( n = 2 , 675 sperm from 4 male mice ) . Data information : All data are presented in the box plot format . The central band in each box represents the median , boxes indicate the middle quartiles , whiskers extended to the minimum and maximum values . * p < 0 . 05 and * * p < 0 . 01 , determined using Student ' s or Welch t - test .

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C. Representative image of 5-mC immunofluorescence in the sperm of saline- and 5-Aza-treated mice (ST and AT, respectively). Scale bar: 100 μm. D. Quantification analysis of 5-mC immunofluorescence in individual sperm cells from STO (n=1,673 sperm from 4 male mice) and ATO (n=2,675 sperm from 4 male mice).Data information: All data are presented in the box plot format. The central band in each box represents the median, boxes indicate the middle quartiles, whiskers extended to the minimum and maximum values. *p<0.05 and **p<0.01, determined using Student's or Welch t-test.


F . Overall USV number in STO and ATO . Data information : All data are presented in the box plot format . The central band in each box represents the median , boxes indicate the middle quartiles , whiskers extended to the minimum and maximum values . * p < 0 . 05 and * * p < 0 . 01 , determined using Student ' s or Welch t - test .

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F. Overall USV number in STO and ATO. Data information: All data are presented in the box plot format. The central band in each box represents the median, boxes indicate the middle quartiles, whiskers extended to the minimum and maximum values. *p<0.05 and **p<0.01, determined using Student's or Welch t-test.


Growth 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 .

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Growth 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 .

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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 .

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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 .

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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 .

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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 .

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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 .

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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 .

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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 ) .

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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 .

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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 .

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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 .

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

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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 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 .

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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 .

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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 .

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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 .

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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 .

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

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 cells treated with TGFβ ( 2 ng / mL for all experiments ) or mock for 48h in the presence of 0 . 5 % FBS .

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

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

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

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


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

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

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


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

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

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


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

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

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


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

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

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

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


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

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

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


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

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

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


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

O O O 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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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

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

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


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

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

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


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

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

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


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

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

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

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


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

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

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


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

O O O O O O O O O O O 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

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


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

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

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


( 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 ) .

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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 ) 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 ) .

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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 ) 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 ) .

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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 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 .

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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 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 .

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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 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 ● .

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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 ) 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 ) .

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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 ) 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 .

(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 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 .

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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 ) 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 .

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

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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 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 ' .

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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 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 ' .

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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 ) 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 ' .

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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 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 ) .

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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 the experimental setup of neuronal differentiation and timelapse imaging under treatment with the 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 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 ) .

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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).


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 ) .

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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).


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 ) .

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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 images of GFP and 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 + cells in the VZ ( yellow circle ) and 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 the electroporation site ( white arrowheads ) indicates loss of apical belt integrity and 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 the nodule , which is surrounded by thickened GFP + processes ( pink arrows ) , Arl13b staining shows the 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 the electroporation site ( white arrowheads ) indicates loss of apical belt integrity and 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 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 ) .

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, 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 .

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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 images of 60d old CTRL and 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 the absence of ECE2 mRNA in 60d old ECE2 KO COs ( pink dotted line indicates position of the 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 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 ) .

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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 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 ) .

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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 + 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 ) .

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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).


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 ) .

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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 - 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 ) .

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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).


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 ) .

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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).


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 ) .

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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).