( C - D ) RT - PCR of expression of miR - 181a ( - 5p - 2 - 3p ) in γδ T isolated from thymus ( C ) or from PBLs ( D ) cultured with indicated cytokines , respectively for 11 days ( C ) or for 4 - 6 days ( D ) . When indicated , γδ PBLs were also co - cultured with an anti - TGF - β blocking antibody . Data are normalized to value obtained in IL - 7 cultures ( n = 4 - 10 independent biological samples ) .

O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O B-CELL_TYPE I-CELL_TYPE O O B-TISSUE O O O O O B-CELL_TYPE O O O O O 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-CELL_TYPE O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O

(C-D) RT-PCR analysis of the expression of miR-181a(-5p and -2-3p) in γδ T cells isolated from thymus (C) or from PBLs (D) and cultured with the indicated cytokines, respectively for 11 days (C) or for 4-6 days (D). When indicated, γδ PBLs were also co-cultured with an anti-TGF-β blocking antibody. Data are normalized to the value obtained in IL-7 cultures (n=4-10 independent biological samples).


( E - F ) RT - PCR of expression of miR - 181a ( - 5p - 2 - 3p ) versus FACS of expression of TNF - α ( left panel ) , IFN - γ ( middle panel ) NKGD2 ( right panel ) in γδ T isolated from thymus ( E ) PBLs ( F ) . Samples were either freshly isolated or cultured for 11 days ( γδ thymocytes ) or for 6 days ( γδ PBLs ) with indicated cytokines ( n = 5 independent biological samples ) .

O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O B-EXP_ASSAY O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-GENEPROD O O O O O B-CELL_TYPE I-CELL_TYPE O O B-TISSUE O O O B-CELL_TYPE O O O O O O O O O O O O O O O B-CELL_TYPE I-CELL_TYPE O O O O O O B-CELL_TYPE I-CELL_TYPE O O O O O O O O O O O O O

(E-F) RT-PCR analysis of the expression of miR-181a(-5p and -2-3p) versus FACS analysis of the expression of TNF-α (left panel), IFN-γ (middle panel) and NKGD2 (right panel) in γδ T cells isolated from thymus (E) and PBLs (F). Samples were either freshly isolated or cultured for 11 days (γδ thymocytes) or for 6 days (γδ PBLs) with the indicated cytokines (n=5 independent biological samples).


( D ) FACS of expression of Annexin V in miR - 181a versus empty transduced ( GFP + ) untransduced ( GFP - ) γδ thymocytes , cultured with IL - 7 plus IL - 2 for 11 days ( n = 5 independent biological samples ) .

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(D) FACS analysis of the expression of Annexin V in miR-181a versus empty transduced (GFP+) and untransduced (GFP-) γδ thymocytes, cultured with IL-7 plus IL-2 for 11 days (n=5 independent biological samples).


( E ) RT - PCR of expression of Bcl2 Bcl - xL ( normalized to values obtained with empty ) in transduced ( GFP + ) γδ thymocytes , cultured with IL - 7 plus IL - 2 for 11 days ( n = 5 independent biological samples ) .

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O B-GENEPROD O O B-CELL_TYPE I-CELL_TYPE O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O

(E) RT-PCR analysis of the expression of Bcl2 and Bcl-xL (normalized to the values obtained with the empty virus) in transduced (GFP+) γδ thymocytes, cultured with IL-7 plus IL-2 for 11 days (n=5 independent biological samples).


( F ) FACS - derived expression of indicated surface intracellular markers in miR - 181a versus empty transduced ( GFP + ) untransduced ( GFP - ) γδ T thymocytes , cultured with IL - 7 plus IL - 2 for 11 days ( n = 7 - 11 independent biological samples ) .

O O O B-EXP_ASSAY O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-GENEPROD O O O O B-GENEPROD O O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O

(F) FACS-derived expression of indicated surface and intracellular markers in miR-181a versus empty transduced (GFP+) and untransduced (GFP-) γδ T thymocytes, cultured with IL-7 plus IL-2 for 11 days (n=7-11 independent biological samples).


( B ) RT - PCR of expression of indicated genes associated with Mapk , Notch , Irf4 or Stat1 signaling in sorted GFP + γδ thymocytes transduced with empty or miR - 181a viral vector . Data are normalized to value obtained with empty . Samples were cultured with IL - 7 plus IL - 2 for 11 days ( n = 3 - 5 independent biological samples ) .

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-CELL_TYPE I-CELL_TYPE O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O

(B) RT-PCR analysis of the expression of the indicated genes associated with Mapk, Notch, Irf4 or Stat1 signaling in sorted GFP+ γδ thymocytes transduced with empty or miR-181a viral vector. Data are normalized to the value obtained with the empty virus. Samples were cultured with IL-7 plus IL-2 for 11 days (n=3-5 independent biological samples).


( C ) Dual luciferase reporter performed to verify binding between miR - 181a Map3k2 , Notch2 , Irf4 or Stat1 mRNA . HEK293 T were co - transfected with a pmirGLO Dual - Luciferase miRNA Target Expression Vector containing either WT or mutated ( Mut ) 3 ′ UTR target sites plus miR - 181a . A negative construct ( without miR - 181a binding sites ) was included . Firefly / Renilla ratios were normalized to those obtained for empty vector ( n = 4 independent biological samples ) .

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O B-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O B-CELL_LINE B-CELL_TYPE O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O

(C) Dual luciferase reporter assay performed to verify binding between miR-181a and Map3k2, Notch2, Irf4 or Stat1 mRNA. HEK293 T cells were co-transfected with a pmirGLO Dual-Luciferase miRNA Target Expression Vector containing either the WT or mutated (Mut) 3′ UTR target sites plus miR-181a. A negative construct (without miR-181a binding sites) was included. Firefly/Renilla ratios were normalized to those obtained for the empty vector (n=4 independent biological samples).


( E - G ) RT - PCR in circulating γδ T of Map3k2 or Notch2 expression ( E ) , Nkg2d or Tnfa expression ( F ) , NKG2D or TNF - α expression ( G ) after siRNA - mediated knockdown of Map3k2 or Notch2 . In each experiment , a control condition was also used where a nontargeting negative control siRNA was transfected . γδ were cultured with IL - 7 for 4 days ( n = 6 independent biological samples ) .

O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-CELL_TYPE I-CELL_TYPE O B-GENEPROD O B-GENEPROD O O O O O B-GENEPROD O B-GENEPROD O O O O O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD B-EXP_ASSAY O O O O O O O O O B-GENEPROD O B-GENEPROD 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-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O

(E-G) RT-PCR analysis in circulating γδ T cells of Map3k2 or Notch2 expression (E), Nkg2d or Tnfa expression (F), and NKG2D or TNF-α protein expression (G) after siRNA-mediated knockdown of Map3k2 or Notch2. In each experiment, a control condition was also used where a nontargeting negative control siRNA was transfected. γδ were cultured with IL-7 for 4 days (n=6 independent biological samples).


( B ) TDP - 43 immunostaining is elevated not only in poly - GA transduced neurons , but also in non - transduced receiver . red arrows indicate with TDP - 43 in GFP positive negative , respectively .

O O O B-GENEPROD I-GENEPROD I-GENEPROD B-EXP_ASSAY O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-CELL_TYPE O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O O O O

(B) Cytoplasmic TDP-43 immunostaining is elevated not only in poly-GA transduced neurons, but also in the non-transduced receiver cells. White and red arrows indicate cells with cytoplasmic TDP-43 in GFP positive and negative cells, respectively.


( C ) Automated of with TDP - 43 in GFP or GA175 - GFP transduced ( donor ) , non - transduced ( receiver ) neurons . with without GFP signal were counted separately ( indicated by + / − ) . Two groups ( GFP negative donor , GFP positive receiver ) were excluded due to very high GFP transduction rate very low GFP transmission rate . n = 4 biological replicates . In total 283 donor GFP , 273 donor GA175 - GFP , 284 receiver GFP 266 receiver GA175 - GFP were analyzed . Scatter plot with bar - graphs of mean ± SD . One - way ANOVA with Tukey ' s multiple comparisons . * * * denotes p < 0 . 001 .

O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O O O O B-CELL_TYPE O O O B-GENEPROD O O O O O O O O O O O O O O O O O B-GENEPROD O O O B-GENEPROD O O O O O O O O O B-GENEPROD O O O O B-GENEPROD O O O O O O O O O O O O O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(C) Automated quantification of cells with cytoplasmic TDP-43 in GFP or GA175-GFP transduced (donor), non-transduced (receiver) neurons. Cells with and without GFP signal were counted separately (indicated by +/−). Two groups (GFP negative donor, GFP positive receiver) were excluded due to very high GFP transduction rate and very low GFP transmission rate. n=4 biological replicates. In total 283 donor GFP, 273 donor GA175-GFP, 284 receiver GFP and 266 receiver GA175-GFP cells were analyzed. Scatter plot with bar-graphs of mean ± SD. One-way ANOVA with Tukey's multiple comparisons test. *** denotes p<0.001.


Co - culture model in HeLa transfected with iRFP or GA175 - iRFP in donor compartment TDP - 43ΔNLS - GFP in donor receiver compartment . Immunofluorescence of TDP - 43 ∆ NLS aggregate number per

O O O O O B-CELL_LINE O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O O O O B-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O

Co-culture model in HeLa cells transfected with iRFP or GA175-iRFP in the donor compartment and TDP-43ΔNLS-GFP in donor and receiver compartment. Immunofluorescence staining of TDP-43∆NLS aggregate number per cell


Co - culture model in HeLa transfected with iRFP or GA175 - iRFP in donor compartment TDP - 43ΔNLS - GFP in donor receiver compartment . automatic of TDP - 43 ∆ NLS aggregate number per In ( E ) n = 3 biological replicates with 368 donor iRFP , 251 donor GA175 - iRFP , 430 receiver iRFP 328 GA175 - iRFP were analyzed . with without GFP signal were analyzed separately ( indicated by + / − ) . red arrows indicate with TDP - 43 in GFP positive negative , respectively . Scatter plot with bar - graphs of mean ± SD . One - way ANOVA with Tukey ' s multiple comparisons .

O O O O O B-CELL_LINE O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O

Co-culture model in HeLa cells transfected with iRFP or GA175-iRFP in the donor compartment and TDP-43ΔNLS-GFP in donor and receiver compartment. automatic quantification of TDP-43∆NLS aggregate number per cell In (E) n=3 biological replicates with 368 donor iRFP, 251 donor GA175-iRFP, 430 receiver iRFP and 328 GA175-iRFP cells were analyzed. Cells with and without GFP signal were analyzed separately (indicated by +/−). White and red arrows indicate cells with cytoplasmic TDP-43 in GFP positive and negative cells, respectively. Scatter plot with bar-graphs of mean ± SD. One-way ANOVA with Tukey's multiple comparisons test.


( F ) in addition to filter trap of SDS - insoluble TDP - 43ΔNLS - GFP compared in iRFP or GA175 - iRFP transfected .

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(F) in addition to filter trap assay of SDS-insoluble TDP-43ΔNLS-GFP aggregates compared in iRFP or GA175-iRFP transfected cells.


( G ) GFP mRNA expression were measured by qPCR . were normalized to GAPDH , β - actin β2 - microglobulin mRNA . Bar - graphs of mean ± SD . Unpaired two - t - with Welch ' s correction . * denotes p < 0 . 05 , * * denotes p < 0 . 01 .

O O O B-GENEPROD B-EXP_ASSAY I-EXP_ASSAY O O O B-EXP_ASSAY O O O O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(G) 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. Unpaired two-tailed t-test with Welch's correction. * denotes p<0.05, and ** denotes p<0.01.


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

O O O B-EXP_ASSAY I-EXP_ASSAY O O O B-GENEPROD O O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O B-CELL_TYPE I-CELL_TYPE O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O O O O O O O O O O O O

(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 of with 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 ) very low GFP transmission rate ( GFP - positive receiver with IgG anti - GA ) 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 were analyzed . Scatter plot with bar - graphs of mean ± SD . One - way ANOVA with Tukey ' s multiple comparisons . * * * denotes p < 0 . 001 .

O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O O O O B-CELL_TYPE O O O O O O O O O B-GENEPROD O O O B-GENEPROD O O O O O O B-GENEPROD O O O B-GENEPROD O O O O O O O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O O O O O O O O O O O O B-GENEPROD O O O B-GENEPROD O O O O O B-GENEPROD O O O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O O O O O O O O O O O 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) 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 antibody leftover pre post immunoprecipitation in 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 in conditioned media before after immunodepletion with control IgG 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 . * * denotes p < 0 . 01 , * * * denotes p < 0 . 001 .

O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-EXP_ASSAY O O O O O B-GENEPROD O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(D) 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 proteasome subunit PSMC4 poly - GA inclusions in spinal cord of GA149 - CFP transgenic mouse Scale bar denotes 20 µm .

O B-EXP_ASSAY O B-SUBCELLULAR O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD B-SUBCELLULAR O B-TISSUE I-TISSUE O B-GENEPROD O B-GENEPROD O B-ORGANISM O O O O O O

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 proteasome subunit PSMC4 poly - GA inclusions in cortex of C9orf72 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 transfected with iRFP or GA175 - iRFP in donor compartment an UbG76V - GFP proteostasis reporter in donor receiver compartment ( 48 h ) . ( C ) Separate of both compartments by immunoblot ( D ) immunoblot . For quantitative 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 . Red dashed indicates control ' s expression level . * denotes p < 0 . 05 , * * * denotes p < 0 . 001 .

O O O O O B-CELL_LINE O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O B-SMALL_MOLECULE O B-GENEPROD O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O B-EXP_ASSAY O O O O B-EXP_ASSAY B-SMALL_MOLECULE O B-GENEPROD O O O B-GENEPROD O O O O O O O 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-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O

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 were measured by qPCR . were normalized to GAPDH , β - actin β2 - microglobulin mRNA . Bar - graphs of mean ± SD . n = 3 biological replicates . Unpaired two - t - with Welch ' s correction .

O O O B-GENEPROD B-EXP_ASSAY I-EXP_ASSAY O O O B-EXP_ASSAY O O O O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O

(E) 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 of a UbG76V - GFP reporter incubated 48h with conditioned media from RFP or GA175 - RFP transfected 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 . * denotes p < 0 . 05 . ( G ) Comparisons of corresponding histograms for compensated RFP UbG76V - GFP from one representative experiment that shows specific transmission of GA175 - RFP associated with accumulation of UbG76V - GFP in incubated with GA175 - RFP conditioned media .

O O O O O B-EXP_ASSAY I-EXP_ASSAY O O B-SMALL_MOLECULE O B-GENEPROD O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O B-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-GENEPROD B-SMALL_MOLECULE O B-GENEPROD O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O B-SMALL_MOLECULE O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O

(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.


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

B-CELL_TYPE I-CELL_TYPE O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O O O O B-SMALL_MOLECULE O O O O O O O O O O B-EXP_ASSAY O O B-GENEPROD I-GENEPROD I-GENEPROD O B-CELL_TYPE O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-SMALL_MOLECULE O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-SMALL_MOLECULE O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O B-GENEPROD B-CELL_TYPE O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-CELL_TYPE O O O O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O B-SMALL_MOLECULE O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O

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.


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

B-CELL_TYPE I-CELL_TYPE O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O O O O B-SMALL_MOLECULE O O O O O O O O O O B-EXP_ASSAY O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O B-GENEPROD O B-GENEPROD I-GENEPROD B-EXP_ASSAY O

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.


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

B-CELL_TYPE I-CELL_TYPE O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O O O O B-SMALL_MOLECULE O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O B-GENEPROD O B-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O

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

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

O O O O O B-CELL_LINE O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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

B-CELL_LINE O O O O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O B-EXP_ASSAY B-GENEPROD O O O O O B-GENEPROD O O O B-GENEPROD O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O B-GENEPROD O O O O B-GENEPROD I-GENEPROD O B-GENEPROD O O O O O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O

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

B-CELL_LINE O O O O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O B-GENEPROD O O B-EXP_ASSAY I-EXP_ASSAY O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O

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

B-CELL_LINE O O O O O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O O O O B-EXP_ASSAY O O O O O O O O O O O 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-GENEPROD O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O

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

B-CELL_LINE O O O O O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O O O O O O B-GENEPROD O O B-EXP_ASSAY I-EXP_ASSAY O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O

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

B-EXP_ASSAY O B-CELL_LINE O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-SMALL_MOLECULE O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O

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 were measured by qPCR . n = 3 biological replicates . Bar - graphs of mean ± SD . Unpaired two - t - 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 transfected with RFP - 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 were co - transfected with indicated TDP - NLS reporters as well as GFP or GA175 - GFP , 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 of RFP - NLS reporters in GFP positive . Note that K84A K84R block overall import , while K95A K95R allow import but are resistant to inhibition by poly - GA . n = 4 biological replicates . total number of 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 , 638 870 . Scatter plot with bar - graphs of mean ± SD . One - way ANOVA with Tukey ' s multiple comparisons . * * * denotes p < 0 . 001 . Scale bar denotes 20 µm .

O O O O O B-GENEPROD O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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

O O O O O B-CELL_LINE O O O O O O B-GENEPROD O B-GENEPROD O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O B-EXP_ASSAY O O O B-GENEPROD B-EXP_ASSAY O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O O O O O O B-GENEPROD O O O B-GENEPROD O O O O O O B-GENEPROD B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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

B-CELL_LINE O O O O O O O O B-GENEPROD O B-GENEPROD O O B-GENEPROD O B-SMALL_MOLECULE B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O O O O O O O O B-EXP_ASSAY O O O O O O O B-GENEPROD O O O O O B-EXP_ASSAY O O O O O O O O B-GENEPROD B-EXP_ASSAY O O O O O O B-GENEPROD O O O O O O O O O O B-GENEPROD O B-SMALL_MOLECULE O O O B-GENEPROD O O O O O O B-GENEPROD B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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

B-CELL_LINE O O O O O O O O B-GENEPROD O B-GENEPROD O O B-GENEPROD O B-SMALL_MOLECULE B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O O O O O O O O B-EXP_ASSAY O O O O O O O B-GENEPROD O O

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 presence of AMP - PNP aRF1 / aPelota . 30S population contains a stoichiometric of ABCE1 ribosomal , forming post - splitting . rps : small subunit ribosomal .

B-GENEPROD O O B-SUBCELLULAR I-SUBCELLULAR O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE B-GENEPROD O B-GENEPROD O B-SUBCELLULAR O O O O O B-GENEPROD B-SUBCELLULAR O O O O O O O O O O B-SUBCELLULAR O

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 post - SC show a growth defect when exchanged for a small one ( alanine ) or a negative charge ( glutamate ) . + + no effect , + growth defect , - lethal .

B-ORGANISM B-EXP_ASSAY O B-GENEPROD O O B-ORGANISM I-ORGANISM I-ORGANISM O O B-ORGANISM I-ORGANISM I-ORGANISM O O O O O O O O B-SUBCELLULAR O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O B-SMALL_MOLECULE O O O O O O B-SMALL_MOLECULE O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O

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 α - helices of hinge 2 prevent 30S binding while Y592A / Y593A ( C - terminus ) 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 S580E display strongly impaired splitting activity . Unspecific ribosome dissociation level as determined in control experiments in absence of ABCE1 is marked by dotted . ATP turnover per ABCE1 is not affected in tested mutants .

B-SUBCELLULAR B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O B-SUBCELLULAR O O O O O O O O O O B-GENEPROD O O O O O B-SMALL_MOLECULE O O B-GENEPROD O O O O O O O

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

O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

C. 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 16 hyper - 96 hypo - methylated DMRs in sperm from aged mice ordered by their chromosomal positions . M indicates 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 embryonic brain of YFO AFO at E14 . 5 ( n = 3 in each group ) . Green lines indicate a twofold change p = 0 . 01 . Orange dots indicate / NRSF target genes . Data information : Data are presented in volcano plot or chart format . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , determined by nominal GSEA p - value .

O O O O O O O B-TISSUE I-TISSUE O O O O O O O O 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-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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

O O O O O O O B-TISSUE I-TISSUE O O O O O O O O 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-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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

O O B-EXP_ASSAY O B-TISSUE I-TISSUE O O O B-TISSUE O O O O O O O O O O O O O B-GENEPROD O O B-GENEPROD O O B-TISSUE I-TISSUE O O O B-GENEPROD O B-TISSUE I-TISSUE O B-GENEPROD O O B-GENEPROD O O B-TISSUE I-TISSUE O O O O O O O O O B-EXP_ASSAY O O B-TISSUE I-TISSUE O O B-TISSUE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

C. 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 postnatal brain of YFO AFO ( representative magnified of boxes in panel A ) . Tbr1 - / Ctip2 - : layer 2 - 4 , Ctip2high : layer 5 , Tbr1 + / Ctip2 - : layer 6 . Scale bar : 100 μm . G . density in each cortical layer in postnatal brain of YFO AFO . Data information : data are presented as mean ± . * p < 0 . 05 , * * p < 0 . 01 , determined using Student t - .

O O B-EXP_ASSAY O O B-TISSUE I-TISSUE O O O B-TISSUE O O O O O O O O O O O O O B-GENEPROD O O B-GENEPROD O O B-TISSUE I-TISSUE O O O B-GENEPROD O B-TISSUE I-TISSUE O B-GENEPROD O O B-GENEPROD O O B-TISSUE I-TISSUE O O O O O O O O O B-EXP_ASSAY O O B-TISSUE I-TISSUE O O B-TISSUE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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

O O O B-EXP_ASSAY O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE B-EXP_ASSAY O B-CELL_TYPE O B-SMALL_MOLECULE O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O B-ORGANISM O O B-SMALL_MOLECULE O O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE B-EXP_ASSAY O B-CELL_TYPE O O O O O O O O B-CELL_TYPE O O B-ORGANISM O B-SMALL_MOLECULE O O O O O O B-CELL_TYPE O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

C. 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 ATO . Data information : data are presented in box plot format . central band in each box represents median , boxes indicate middle quartiles , whiskers extended to minimum maximum values . * p < 0 . 05 * * p < 0 . 01 , determined using Student ' s or Welch t - .

O O O B-EXP_ASSAY I-EXP_ASSAY O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

F. 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 treated with TGFβ ( 2 ng / mL for experiments ) or vehicle alone ( mock ) for indicated time . number at indicated days relative to number at start of treatment ( d0 ) is shown . dotted represents 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 were treated with TGFβ or mock for 48h incubated with O - propargyl - puromycin ( OPP ) for last 60 min . translation rate was determined by flow cytometry for OPP incorporation into . A representative plot is shown on left , is shown on right . Values are relative to mock - treated .

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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 treated with TGFβ or mock for 48h .

Western blot of NIH-3T3 cells treated with TGFβ or mock for 48h.


Western blot of ECM from NIH - 3T3 grown in presence of absence of TGFβ of lysates from NIH - 3T3 grown on a parallel plate . Note absence of tubulin 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 according to ( d ) , collagen abundance was measured by picrosirius red , normalized to packed volume before ECM extraction , expressed relative to mock - treated .

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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 were treated with TGFβ or mock for 48h . medium was replaced in last 12h of treatment ( with same treatments as before ) , glucose consumption from fresh media were measured using 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 ) mitochondrial oxidative phosphorylation ( JATPox ) from NIH - 3T3 treated with TGFβ or mock for 48h , calculated using Seahorse data shown in ( k ) 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 ) OPP incorporation of NIH - 3T3 treated with TGFβ or mock for 2h , 4h , 8h , 24h 48h , relative to mock - treated . Also see EV Figure 1k l . Note that datapoints of 2h 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 were treated with TGFβ or mock for 48h . medium was replaced in last 12h of treatment ( with 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 were treated with TGFβ or mock for 48h , oxygen consumption rate ( OCR ) before after treatment with mitochondrial inhibitors was measured using Seahorse bioanalyzer . Oligo , Oligomycin ; Rot / Anti - A , Rotenone / Antimycin - A ; PCV , packed 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 were treated with TGFβ or mock for 48h reduced ( GSH ) oxidized ( GSSG ) glutathione were measured by LC - MS / MS . Shown is GSH / GSSG .

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


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

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

O O B-SMALL_MOLECULE O O B-ORGANISM B-SMALL_MOLECULE O O O O O O O O O O O

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


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

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

O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O B-EXP_ASSAY O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O

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


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

B-EXP_ASSAY I-EXP_ASSAY O B-SMALL_MOLECULE O B-SMALL_MOLECULE I-SMALL_MOLECULE O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O B-GENEPROD O O O O O O O O O O O

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


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

O O O B-EXP_ASSAY O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O B-GENEPROD O O O O O O O O O O O B-EXP_ASSAY O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O

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


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

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

O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O B-GENEPROD O O B-SUBCELLULAR O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O B-GENEPROD O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

O O B-EXP_ASSAY I-EXP_ASSAY O O B-EXP_ASSAY O B-GENEPROD O O O O B-TISSUE O O B-ORGANISM O B-DISEASE I-DISEASE O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O

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


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

O O O O O O O O B-TISSUE O O B-DISEASE I-DISEASE I-DISEASE O B-DISEASE O O O O O O O O O O O O O O O O O O O O O

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


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

O O O B-GENEPROD B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O

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


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

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


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

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


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

O O B-EXP_ASSAY I-EXP_ASSAY O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O B-GENEPROD O O O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O O O O B-SMALL_MOLECULE O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O

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


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

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


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

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


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

B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O B-GENEPROD O O O O O B-SMALL_MOLECULE O O O O O O O O O O O B-SMALL_MOLECULE O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O

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


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

B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O B-GENEPROD O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O

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


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

B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O B-GENEPROD O O O O O O O O O O O B-SMALL_MOLECULE O O O B-EXP_ASSAY I-EXP_ASSAY O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O B-SMALL_MOLECULE O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O

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


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

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


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

B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O O O O O O O O B-GENEPROD O O O O O B-SMALL_MOLECULE O O O B-EXP_ASSAY I-EXP_ASSAY O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O

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


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

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


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

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


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

B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O O O O B-GENEPROD O O O O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O B-EXP_ASSAY I-EXP_ASSAY O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O

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


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

B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O B-SMALL_MOLECULE O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O B-SMALL_MOLECULE O O O B-EXP_ASSAY I-EXP_ASSAY O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O

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


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

O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O O O O O O O O B-GENEPROD O O O O O B-SMALL_MOLECULE O O O B-EXP_ASSAY I-EXP_ASSAY O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O O O O O O

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


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

B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O O O O O O O O B-GENEPROD O O O O B-SMALL_MOLECULE O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O

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


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

B-EXP_ASSAY I-EXP_ASSAY O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O O O O O O O B-GENEPROD O O O O O

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


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

B-SMALL_MOLECULE O O B-SUBCELLULAR O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O O O O O O O O O O O B-GENEPROD O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O

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


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

B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O B-GENEPROD O O O O B-GENEPROD O O O O O B-SMALL_MOLECULE O O O B-EXP_ASSAY I-EXP_ASSAY O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O

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


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

B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O B-GENEPROD O O O O B-GENEPROD O O O O O B-SMALL_MOLECULE O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O

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


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

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


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

B-SMALL_MOLECULE O O B-SUBCELLULAR O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O B-GENEPROD O O O O O O O B-GENEPROD O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O

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


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

O O B-EXP_ASSAY I-EXP_ASSAY O O B-EXP_ASSAY O B-GENEPROD O O O O O O O O O O B-TISSUE O O B-ORGANISM O B-DISEASE I-DISEASE O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O

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


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

O O B-EXP_ASSAY I-EXP_ASSAY O O B-EXP_ASSAY O B-GENEPROD O O O O O O O O O O O O O O O B-TISSUE O O B-DISEASE I-DISEASE I-DISEASE O B-DISEASE O O O O O O O O O O O O O O O O O O O O O

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


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

O O O B-GENEPROD I-GENEPROD B-EXP_ASSAY O B-DISEASE O O O B-DISEASE O O O O O B-GENEPROD O B-GENEPROD I-GENEPROD O B-GENEPROD O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD I-GENEPROD O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O 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 ) Δapg10 ( TFD10 ‐ L1 ) were incubated in SD ( − N ) medium containing 1 mM PMSF at 30 ° C . After incubation for 6 h , were observed under microscopy ( Nomarski ) .

O O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM O O B-GENEPROD O O O O B-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM O O O B-ORGANISM I-ORGANISM I-ORGANISM O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O B-EXP_ASSAY O O O O

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


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

O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O 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-EXP_ASSAY O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O

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


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

O O O O O O O O O O O B-GENEPROD O O O O O B-GENEPROD O O O O O O B-GENEPROD O O O O O O O O B-GENEPROD O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O B-GENEPROD O O O O O

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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

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


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

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