( I ) Double immunofluorescence labeling using antibodies against DYRK3 and FUS in lumbar spinal cord α - MNs of healthy subjects ( control ) and fALS patients carrying the p . 521C mutation in the FUS gene . Control ( upper panel ) : α - MNs showing uniform cytoplasmic ( white arrows ) and speckled pattern of strong nuclear immunoreactivity ( white arrowhead ) of DYRK3 , as well as diffuse nuclear FUS immunoreactivity . fALS ( p . R521C - FUS ; lower panel ) : surviving α - MNs harboring large FUS aggregates ( yellow arrows ) showed markedly reduced DYRK3 immunoreactivity both in the cytoplasm as well as in the nucleus ( red arrowheads ) . Instead , α - MNs devoid of FUS aggregates ( white arrows ) showed normal DYRK3 immunoreactivity similar to the one of α - MNs from normal controls . Asterix ( * ) represents non - specific lipofuscin granules in one of the α - MN in FUS - ALS . Paraffin sections ; scale bars is 50 µm . ( J ) Quantification of α - MNs showing low or absent DYRK3 staining ( upper graphic ) and FUS aggregates ( lower graphic ) in lumbar spinal cord from five healthy subjects ( control ) and five fALS patients carrying the p . 521C mutation in the FUS gene . Total number of α - MNs analyzed : 403 ( control ) ; 132 ( fALS ) . n = 5 , ± sem ( Student ' s t - test ) .

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(I) Double immunofluorescence labeling using antibodies against DYRK3 and FUS in lumbar spinal cord α-MNs of healthy subjects (control) and fALS patients carrying the p.521C mutation in the FUS gene. Control (upper panel): α-MNs showing uniform cytoplasmic (white arrows) and speckled pattern of strong nuclear immunoreactivity (white arrowhead) of DYRK3, as well as diffuse nuclear FUS immunoreactivity. fALS (p.R521C-FUS; lower panel): surviving α-MNs harboring large FUS aggregates (yellow arrows) showed markedly reduced DYRK3 immunoreactivity both in the cytoplasm as well as in the nucleus (red arrowheads). Instead, α-MNs devoid of FUS aggregates (white arrows) showed normal DYRK3 immunoreactivity similar to the one of α-MNs from normal controls. Asterix (*) represents non-specific lipofuscin granules in one of the α-MN in FUS-ALS. Paraffin sections; scale bars is 50 µm. (J) Quantification of α-MNs showing low or absent DYRK3 staining (upper graphic) and FUS aggregates (lower graphic) in lumbar spinal cord from five healthy subjects (control) and five fALS patients carrying the p.521C mutation in the FUS gene. Total number of α-MNs analyzed: 403 (control); 132 (fALS). n = 5, ± sem (Student's t-test).


A HeLa cells were transfected with control ( Ctr ) or three different GCN2 - targeting siRNA - s and progression from prophase to anaphase was observed by live - cell imaging . Averages ( lines ) and SEM ( bands ) from five ( siCtr ) or four ( each siGCN2 ) independent experiments are shown ; n = 379 for siCtr , 241 for siGCN2 # 1 , 170 for siGCN2 # 2 , 239 for siGCN2 # 3 . * * * * p < 0 . 0001 for each of the siRNA - s ( Kolmogorov - Smirnov test ) . t = 0 denotes entry into prophase as judged by the first frame showing chromatin condensation .

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A HeLa cells were transfected with control (Ctr) or three different GCN2-targeting siRNA-s and progression from prophase to anaphase was observed by live-cell imaging. Averages (lines) and SEM (bands) from five (siCtr) or four (each siGCN2) independent experiments are shown; n= 379 for siCtr, 241 for siGCN2 #1, 170 for siGCN2 #2, 239 for siGCN2 #3. ****p<0.0001 for each of the siRNA-s (Kolmogorov-Smirnov test). t=0 denotes entry into prophase as judged by the first frame showing chromatin condensation.


E , Chromosome congression is affected by the absence of GCN2 . E Immunofluorescence images of HeLa cells stained for a kinetochore marker , CREST ( grey ) and DAPI for DNA ( blue ) . Arrowheads indicate misaligned chromosomes . Scale bars represent 5 µm .

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E, Chromosome congression is affected by the absence of GCN2. E Immunofluorescence images of HeLa cells stained for a kinetochore marker, CREST (grey) and DAPI for DNA (blue). Arrowheads indicate misaligned chromosomes. Scale bars represent 5 µm.


G Increased number of mitotic cells with tripolar spindles after GCN2 knock - down . HeLa cells were transfected with control ( Ctr ) or GCN2 - targeting siRNA and fixed for immunofluorescence . The number of spindle poles was determined by using a pericentrin antibody . Mean and SEM are shown from five independent experiments . n = 61 , 27 , 19 , 24 and 28 for siCtr ; n = 49 , 29 , 22 , 26 and 26 for siGCN2 ; p = 0 . 0125 , paired two - tailed t - test .

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G Increased number of mitotic cells with tripolar spindles after GCN2 knock-down. HeLa cells were transfected with control (Ctr) or GCN2-targeting siRNA and fixed for immunofluorescence. The number of spindle poles was determined by using a pericentrin antibody. Mean and SEM are shown from five independent experiments. n= 61, 27, 19, 24 and 28 for siCtr; n=49, 29, 22, 26 and 26 for siGCN2; p=0.0125, paired two-tailed t-test.


A , B There is no significant difference in global translation rates in mitosis after GCN2 knock - down . A HeLa cells were pulse labeled with OPP and fixed with 70 % ethanol . OPP was detected by Click - IT ( AF 647 ) and mitotic cells were identified by staining for phospho - histone H3 . 2D flow cytometry plots are shown , where AF647 intensity corresponds to OPP incorporation . Translation levels normalized to siCtr G1 . The graph shows averages and SEM from three independent experiments . Paired two - tailed t - tests .

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A, B There is no significant difference in global translation rates in mitosis after GCN2 knock-down. A HeLa cells were pulse labeled with OPP and fixed with 70% ethanol. OPP was detected by Click-IT (AF 647) and mitotic cells were identified by staining for phospho-histone H3. 2D flow cytometry plots are shown, where AF647 intensity corresponds to OPP incorporation. Translation levels normalized to siCtr G1. The graph shows averages and SEM from three independent experiments. Paired two-tailed t-tests.


D , Mitotic translation of selected proteins . Hela cells were incubated with 5 µM Eg5i overnight and mitotic cells were shaken off . MG132 was added to 50 µM ( t = 0 ) , then after 10 minutes the cells were split in two ( t = 10 ) . GCN2i ( Gcn2 - IN - 1 , MedChem Express HY - 100877 ) was added to 2 µM to one of the cell suspensions and samples were taken for immunoblotting every 10 minutes ( D ) Representative immunoblots of three biological replicates are shown .

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D, Mitotic translation of selected proteins. Hela cells were incubated with 5 µM Eg5i overnight and mitotic cells were shaken off. MG132 was added to 50 µM (t=0), then after 10 minutes the cells were split in two (t=10). GCN2i (Gcn2-IN-1, MedChem Express HY-100877) was added to 2 µM to one of the cell suspensions and samples were taken for immunoblotting every 10 minutes (D) Representative immunoblots of three biological replicates are shown.


E Mitotic translation of selected proteins . Hela cells were incubated with 5 µM Eg5i overnight and mitotic cells were shaken off . MG132 was added to 50 µM ( t = 0 ) , then after 10 minutes the cells were split in two ( t = 10 ) . GCN2i ( Gcn2 - IN - 1 , MedChem Express HY - 100877 ) was added to 2 µM to one of the cell suspensions and samples were taken for immunoblotting every 10 minutes Representative ( E ) quantifications of three biological replicates ; averages and SEM are shown . Values are normalized to t = 0 .

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E Mitotic translation of selected proteins. Hela cells were incubated with 5 µM Eg5i overnight and mitotic cells were shaken off. MG132 was added to 50 µM (t=0), then after 10 minutes the cells were split in two (t=10). GCN2i (Gcn2-IN-1, MedChem Express HY-100877) was added to 2 µM to one of the cell suspensions and samples were taken for immunoblotting every 10 minutes Representative (E) quantifications of three biological replicates; averages and SEM are shown. Values are normalized to t=0.


A Verification of the mutants . HeLa cells transduced with the indicated GCN2 - siR constructs were transfected with control ( Ctr ) or GCN2 - targeting siRNA . Halofuginone ( HFG ) was added to 40 nM for 4 hours , and GCN2 autophosphorylation , ATF4 induction and eIF2α phosphorylation were assessed by immunoblotting . γ - tubulin is shown as loading control . The line in the GCN2 blot indicates that a longer exposure of the same blot is shown on the left than on the right .

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A Verification of the mutants. HeLa cells transduced with the indicated GCN2-siR constructs were transfected with control (Ctr) or GCN2-targeting siRNA. Halofuginone (HFG) was added to 40 nM for 4 hours, and GCN2 autophosphorylation, ATF4 induction and eIF2α phosphorylation were assessed by immunoblotting. γ-tubulin is shown as loading control. The line in the GCN2 blot indicates that a longer exposure of the same blot is shown on the left than on the right.


C , GCN2 kinase activity is regulated in the cell cycle . HeLa cells were arrested at the G1 / S transition using thymidine and released from the cell cycle at time 0 h . Samples were taken at the indicated time - points for immunoblotting Aurora A and Aurora A - P are shown to indicate the timing of mitosis , GCN2 - P is shown to assess GCN2 activity . Representative immunoblot of GCN2 phosphorylation normalized to GCN2 ( total ) , three biological repeats , three technical repeats each .

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C, GCN2 kinase activity is regulated in the cell cycle. HeLa cells were arrested at the G1/S transition using thymidine and released from the cell cycle at time 0 h. Samples were taken at the indicated time-points for immunoblotting Aurora A and Aurora A-P are shown to indicate the timing of mitosis, GCN2-P is shown to assess GCN2 activity. Representative immunoblot of GCN2 phosphorylation normalized to GCN2 (total), three biological repeats, three technical repeats each.


B GCN2 coimmunoprecipitates with PP1α and γ . HeLa cells stably transfected with GFP - PP1α or GFP - PP1γ were transduced to express either GCN2 - siR ( wild - type ) or GCN2 - siR - AAXA . Cells were arrested in prometaphase with Eg5i , GFP - PP1α and GFP - PP1γ were immunoprecipitated using GFP - trap and the immunoprecipitates were immunoblotted for GCN2 . The vertical line in the PP1α and PP1γ blots indicates that different exposures of the same blot are shown for the inputs and the IP - s .

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B GCN2 coimmunoprecipitates with PP1α and γ. HeLa cells stably transfected with GFP-PP1α or GFP-PP1γ were transduced to express either GCN2-siR (wild-type) or GCN2-siR-AAXA. Cells were arrested in prometaphase with Eg5i, GFP-PP1α and GFP-PP1γ were immunoprecipitated using GFP-trap and the immunoprecipitates were immunoblotted for GCN2. The vertical line in the PP1α and PP1γ blots indicates that different exposures of the same blot are shown for the inputs and the IP-s.


D GCN2 can phosphorylate PP1α and γ in vitro . HeLa cells carrying GFP - PP1α or GFP - PP1γ were arrested in prometaphase with an Eg5 inhibitor ( Lane 3 ) , then GFP - tagged PP1α or PP1γ were immunoprecipitated using GFP - trap ( input , Lane 2 ) . The immunoprecipitates were incubated in kinase buffer and ATP with constitutively active , recombinant GCN2 . The samples were run on phos - tag gels and immunoblotted with PP1γ or PP1α antibody .

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D GCN2 can phosphorylate PP1α and γ in vitro. HeLa cells carrying GFP-PP1α or GFP-PP1γ were arrested in prometaphase with an Eg5 inhibitor (Lane 3), then GFP-tagged PP1α or PP1γ were immunoprecipitated using GFP-trap (input, Lane 2). The immunoprecipitates were incubated in kinase buffer and ATP with constitutively active, recombinant GCN2. The samples were run on phos-tag gels and immunoblotted with PP1γ or PP1α antibody.


F PP1α and PP1γ bandshifts are diminished in cells depleted for GCN2 and arrested in mitosis . HeLa cells were transfected with control or GCN2 - targeting siRNA and arrested in prometaphase using a PLK1 inhibitor . Representative quantifications of the ratios of the phospho - PP1 / PP1 signal ( average and SEM ) from four independent experiments are shown . * p = 0 . 0217 ( PP1α ) and * * p = 0 . 0069 ( PP1γ ) ( paired two - tailed t - tests ) .

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F PP1α and PP1γ bandshifts are diminished in cells depleted for GCN2 and arrested in mitosis. HeLa cells were transfected with control or GCN2-targeting siRNA and arrested in prometaphase using a PLK1 inhibitor. Representative quantifications of the ratios of the phospho-PP1/PP1 signal (average and SEM) from four independent experiments are shown. *p=0.0217 (PP1α) and **p=0.0069 (PP1γ) (paired two-tailed t-tests).


HeLa cells were transfected with control or GCN2 - targeting siRNA and fixed for immunofluorescence . Representative image of mitotic cells stained for A ChTOG ( magenta ) , tubulin ( green ) and DNA ( blue ) , Scale bars represent 5 µm . Percentage of cells displaying the aberrant localizations Averages and SEM from three independent experiments are shown . For chTOG n = 29 , 37 , 44 for siCtr ; n = 14 , 41 , 28 for siGCN2 ; * * p = 0 . 0063 ( paired two - tailed t - tests ) .

HeLa cells were transfected with control or GCN2-targeting siRNA and fixed for immunofluorescence. Representative image of mitotic cells stained for A ChTOG (magenta), tubulin (green) and DNA (blue), Scale bars represent 5 µm. Percentage of cells displaying the aberrant localizations Averages and SEM from three independent experiments are shown. For chTOG n= 29, 37, 44 for siCtr; n= 14, 41, 28 for siGCN2; **p=0.0063 (paired two-tailed t-tests).


HeLa cells were transfected with control or GCN2 - targeting siRNA and fixed for immunofluorescence . Representative image of mitotic cells stained for B CENPE ( magenta ) , P - Aurora B ( yellow ) , tubulin ( green ) and DNA ( blue ) are shown . Scale bars represent 5 µm . D Percentage of cells displaying the aberrant localizations Averages and SEM from three independent experiments are shown . For CENPE n = 15 , 35 , 43 for siCtr ; n = 11 , 21 , 20 for siGCN2 ; * * p = 0 . 001 , for P - Aurora B n = 29 , 37 , 44 for siCtr ; n = 14 , 41 , 28 for siGCN2 ; * * p = 0 . 0053 ( paired two - tailed t - tests ) .

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HeLa cells were transfected with control or GCN2-targeting siRNA and fixed for immunofluorescence. Representative image of mitotic cells stained for B CENPE (magenta), P-Aurora B (yellow), tubulin (green) and DNA (blue) are shown. Scale bars represent 5 µm. D Percentage of cells displaying the aberrant localizations Averages and SEM from three independent experiments are shown. For CENPE n= 15, 35, 43 for siCtr; n= 11, 21, 20 for siGCN2; **p=0.001, for P-Aurora B n=29, 37, 44 for siCtr; n= 14, 41, 28 for siGCN2; **p=0.0053 (paired two-tailed t-tests).


HeLa cells were transfected with control or GCN2 - targeting siRNA , synchronized in the cell cycle by thymidine block - and - release and samples were collected for flow cytometry at the indicated time points . A Cell - cycle progression in the cells synchronized by thymidine block - and - release , three independent experiments , averages and SEM are shown .

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HeLa cells were transfected with control or GCN2-targeting siRNA, synchronized in the cell cycle by thymidine block-and-release and samples were collected for flow cytometry at the indicated time points. A Cell-cycle progression in the cells synchronized by thymidine block-and-release, three independent experiments, averages and SEM are shown.


HeLa cells were transfected with control or GCN2 - targeting siRNA , synchronized in the cell cycle by thymidine block - and - release and samples were collected for immunoblotting at the indicated time points . B Representative immunoblots of TACC3 - S558 phosphorylation and Aurora A - T288

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HeLa cells were transfected with control or GCN2-targeting siRNA, synchronized in the cell cycle by thymidine block-and-release and samples were collected for immunoblotting at the indicated time points. B Representative immunoblots of TACC3-S558 phosphorylation and Aurora A-T288 phosphorylation


F Dephosphorylation of PP1 substrates after Aurora A inhibition in the presence and absence of GCN2 activity . HeLa cells were treated with Aurora Ai ( MLN8237 , MedChemTronica HY - 10971 ) , and with both Aurora Ai and GCN2i . Cell suspensions were incubated at 37oC and at the indicated times samples were removed , mixed with Laemmli sample buffer and boiled . F The immunoblots were probed with the indicated antibodies .

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F Dephosphorylation of PP1 substrates after Aurora A inhibition in the presence and absence of GCN2 activity. HeLa cells were treated with Aurora Ai (MLN8237, MedChemTronica HY-10971), and with both Aurora Ai and GCN2i. Cell suspensions were incubated at 37oC and at the indicated times samples were removed, mixed with Laemmli sample buffer and boiled. F The immunoblots were probed with the indicated antibodies.


B HeLa cells were transfected with control or GCN2 - targeting siRNA or treated with the indicated concentrations of GCN2 inhibitor , cells were fixed after 72 h incubation , and the number of cells containing micronuclei was determined by DAPI staining . Four independent experiments , averages with SD are shown . n = 187 , 72 , 162 , 531 for siCtr ; n = 159 , 112 , 200 , 656 for siGCN2 ; p = 0 . 009 ; n = 721 , 821 , 682 for 0 µM ; n = 607 , 585 , 635 for 1 µM , n = 414 , 510 , 723 for 2 µM ; n = 123 , 147 , 280 for 3 µM ; p = 0 . 004 for 1 µM , * * p = 0 . 005 for 2 µM , * * p = 0 . 005 for 3 µM ( paired two - tailed t - tests ) .

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B HeLa cells were transfected with control or GCN2-targeting siRNA or treated with the indicated concentrations of GCN2 inhibitor, cells were fixed after 72 h incubation, and the number of cells containing micronuclei was determined by DAPI staining. Four independent experiments, averages with SD are shown. n= 187, 72, 162, 531 for siCtr; n= 159, 112, 200, 656 for siGCN2; p= 0.009; n=721, 821, 682 for 0 µM; n= 607, 585, 635 for 1 µM, n= 414, 510, 723 for 2 µM; n= 123, 147, 280 for 3 µM; p=0.004 for 1 µM, **p=0.005 for 2 µM, **p=0.005 for 3 µM (paired two-tailed t-tests).


C Progression from prophase to anaphase in the presence of the GCN2i at the indicated concentrations was observed by live - cell imaging in C hTert - RPE1 cells . Averages ( lines ) and SEM ( bands ) from three ( hTert - RPE1 ) independent experiments are shown t = 0 denotes entry into prophase . for hTert - RPE1 cells n = 276 , 284 , 231 and 228 for 0 , 1 , 2 , and 3 µM GCN2i , respectively . * p = 0 . 0168 ; * * * * < 0 . 0001 ( Kolmogorov - Smirnov test ) .

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C Progression from prophase to anaphase in the presence of the GCN2i at the indicated concentrations was observed by live-cell imaging in C hTert-RPE1 cells. Averages (lines) and SEM (bands) from three (hTert-RPE1) independent experiments are shown t=0 denotes entry into prophase. for hTert-RPE1 cells n= 276, 284, 231 and 228 for 0, 1, 2, and 3 µM GCN2i, respectively. *p=0.0168; ****<0.0001 (Kolmogorov-Smirnov test).


E PP1α and PP1γ bandshifts are diminished in cells arrested in mitosis upon pharmacological inhibition of GCN2 . HeLa or hTert - RPE1 cells were arrested in mitosis using a PLK1 inhibitor and GCN2 - IN1 was added to 2 µM for 3 h before harvesting the cells . Representative E quantifications of the ratios of the phospho - PP1 / PP1 signal ( average and SEM ) from four independent experiments are shown . HeLa : * * * p = 0 . 0007 ( PP1α ) ; * * p = 0 . 0023 ( PP1γ ) ; htert - RPE1 : * p = 0 . 0111 ( PP1α ) and * * * p = 0 . 0007 ( PP1γ ) , paired two - tailed t - tests .

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E PP1α and PP1γ bandshifts are diminished in cells arrested in mitosis upon pharmacological inhibition of GCN2. HeLa or hTert-RPE1 cells were arrested in mitosis using a PLK1 inhibitor and GCN2-IN1 was added to 2 µM for 3 h before harvesting the cells. Representative E quantifications of the ratios of the phospho-PP1/PP1 signal (average and SEM) from four independent experiments are shown. HeLa: ***p=0.0007 (PP1α); **p=0.0023 (PP1γ); htert-RPE1: *p=0.0111 (PP1α) and ***p=0.0007(PP1γ), paired two-tailed t-tests.


Progression from prophase to anaphase in the presence of the Aurora A and GCN2 inhibitors was observed by live - cell imaging in HeLa and hTert - RPE1 cells . GCN2 inhibitor ( " G " ) and Aurora A inhibitor ( " A " ) was added C , D Representative images from experiments shown in A and B , respectively .

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Progression from prophase to anaphase in the presence of the Aurora A and GCN2 inhibitors was observed by live-cell imaging in HeLa and hTert-RPE1 cells. GCN2 inhibitor ("G") and Aurora A inhibitor ("A") was added C, D Representative images from experiments shown in A and B, respectively.


E , F Percentages of cells with micronuclei and mitotic slippage in the experiments shown in A and B . Averages and SEM from three independent experiments are shown . HeLa mitotic slippage * p = 0 . 0137 , HeLa micronculei p = 0 . 0774 , hTert - RPE1 mitotic slippage * * p = 0 . 0011 , hTert - RPE1 micronuclei p = 0 . 0640 ; ( two - tailed t - tests ) .

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E, F Percentages of cells with micronuclei and mitotic slippage in the experiments shown in A and B. Averages and SEM from three independent experiments are shown. HeLa mitotic slippage *p=0.0137, HeLa micronculei p=0.0774, hTert-RPE1 mitotic slippage **p=0.0011, hTert-RPE1 micronuclei p=0.0640; (two-tailed t-tests).


B HeLa and Tert - RPE1 cells stably expressing the cell - cycle markers CDT1 - Ruby and GMNN - Neongreen were treated with the Aurora A and GCN2 inhibitors and cell - cycle progression was followed by live - cell imaging . Representative examples of HeLa and hTert - RPE1 cells are shown from the supplementary videos 4 and 5 . Scale bars represent 5 µM .

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B HeLa and Tert-RPE1 cells stably expressing the cell-cycle markers CDT1-Ruby and GMNN-Neongreen were treated with the Aurora A and GCN2 inhibitors and cell-cycle progression was followed by live-cell imaging. Representative examples of HeLa and hTert-RPE1 cells are shown from the supplementary videos 4 and 5. Scale bars represent 5 µM.


C HeLa and hTert - RPE1 cells were seeded in triplicate and incubated in the presence of the GCN2 and Aurora A inhibitors for 7 ( HeLa ) or 10 ( RPE1 ) days , then fixed and colonies were counted . Shown are averages and SEM from three biological replicates . For HeLa cells * p = 0 . 0267 for A versus G2A ; * p = 0 . 0432 A versus G3A ( paired two - tailed t - tests ) . For hTert - RPE1 cells * p = 0 . 0391 A versus G2A ; * * p = 0 . 021 A versus G3A ( paired two - tailed t - tests ) . D HeLa and Tert - RPE1 cells were treated with the indicated inhibitors for 72 h , stained with Hoechst and analyzed by flow cytometry . The graph shows the percentage of Hoechst - positive ( dead ) cells in four independent experiments . Averages and SEM are shown . For HeLa cells * * p = 0 . 0087 A versus AG ; * * p = 0 . 0027 Ctr versus AG ; * p = 0 . 0237 Ctr versus G ; p = 0 . 0809 Ctr versus A ( paired two - tailed t - test ) . For hTert - RPE1 cells * * * p = 0 . 0005 A versus AG ; * * p = 0 . 0036 Ctr versus AG ; * p = 0 . 0210 Ctr versus G ; p = 0 . 29184 Ctr versus A ( paired two - tailed t - tests ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 HeLa and hTert-RPE1 cells were seeded in triplicate and incubated in the presence of the GCN2 and Aurora A inhibitors for 7 (HeLa) or 10 (RPE1) days, then fixed and colonies were counted. Shown are averages and SEM from three biological replicates. For HeLa cells *p=0.0267 for A versus G2A; *p= 0.0432 A versus G3A (paired two-tailed t-tests). For hTert-RPE1 cells *p=0.0391 A versus G2A; **p=0.021 A versus G3A (paired two-tailed t-tests). D HeLa and Tert-RPE1 cells were treated with the indicated inhibitors for 72 h, stained with Hoechst and analyzed by flow cytometry. The graph shows the percentage of Hoechst-positive (dead) cells in four independent experiments. Averages and SEM are shown. For HeLa cells **p=0.0087 A versus AG; **p=0.0027 Ctr versus AG; *p= 0.0237 Ctr versus G; p= 0.0809 Ctr versus A (paired two-tailed t-test). For hTert-RPE1 cells ***p=0.0005 A versus AG; **p=0.0036 Ctr versus AG; *p=0.0210 Ctr versus G; p= 0.29184 Ctr versus A (paired two-tailed t-tests).


E , F HeLa and Tert - RPE1 cells were treated with the indicated inhibitors for 72 h , stained with the CellEvent ™ Caspase - 3 / 7 green detection reagent and Hoechst and analyzed by flow cytometry . Representative 2D plots of Alexa - Fluor 488 ( a measure of Caspase - 3 / 7 activity ) and Hoechst intensity are shown for E HeLa and F RPE1 cells . All single cells are plotted in magenta , Hoechst - negative cells are overlaid in grey . Note that all Hoechst - positive ( dead ) cells are also stained with the Caspase - 3 / 7 green reagent , indicating apoptosis .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 and Tert-RPE1 cells were treated with the indicated inhibitors for 72 h, stained with the CellEvent™ Caspase-3/7 green detection reagent and Hoechst and analyzed by flow cytometry. Representative 2D plots of Alexa-Fluor 488 (a measure of Caspase-3/7 activity) and Hoechst intensity are shown for E HeLa and F RPE1 cells. All single cells are plotted in magenta, Hoechst-negative cells are overlaid in grey. Note that all Hoechst-positive (dead) cells are also stained with the Caspase-3/7 green reagent, indicating apoptosis.


E and F , Naïve WT CD4 + CD62Lhi T cells were cultured under Th17 polarizing conditions . On day 3 , cells were transfected with control or siRNA targeting Batf , rested overnight , used for total RNA isolation to assess gene expression ( E ) , or restimulated with anti - CD3 for 24 h to measure cytokine production by ELISA ( F ) . Data information : , mean ± s . e . m . of 3 - 5 independent experiments ( * p < 0 . 05 ; two - sided t - test or one - way ANOVA followed by Tukey ' s test ) . RQ , relative quantification .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 and F, Naïve WT CD4+CD62Lhi T cells were cultured under Th17 polarizing conditions. On day 3, cells were transfected with control or siRNA targeting Batf, rested overnight, used for total RNA isolation to assess gene expression (E), or restimulated with anti-CD3 for 24 h to measure cytokine production by ELISA (F). Data information: , mean ± s.e.m. of 3-5 independent experiments (*p<0.05; two-sided t-test or one-way ANOVA followed by Tukey's test). RQ, relative quantification.


Naïve CD4 + CD62Lhi T cells from IL - 17a reporter mice ( Il17aeGFP ) were cultured under Th17 polarizing conditions . On day 5 , eGFP + / IL - 17a + cells were sorted and cultured for two additional rounds in the presence of anti - CD3 alone ( TCR ( R3 ) ) or Th17 polarizing cytokines ( Th17 ( R3 ) ) ( G ) Data information : Data are representative of 3 - 5 independent

Naïve CD4+CD62Lhi T cells from IL-17a reporter mice (Il17aeGFP) were cultured under Th17 polarizing conditions. On day 5, eGFP+/IL-17a+ cells were sorted and cultured for two additional rounds in the presence of anti-CD3 alone (TCR(R3)) or Th17 polarizing cytokines (Th17(R3)) (G) Data information: Data are representative of 3-5 independent experiments


WT and Batf KO mice were orally infected by gavage with 2x109 cfu / ml of Citrobacter rodentium ( Cr ) expressing GFP quantification of the signal ( O ) at the indicated time points . Data information : mean ± s . e . m . of n = 5 - 6 mice ( O ) ( * p < 0 . 05 ; two - sided t - test or one - way ANOVA followed by Tukey ' s test ) .

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

WT and Batf KO mice were orally infected by gavage with 2x109 cfu/ml of Citrobacter rodentium (Cr) expressing GFP quantification of the signal (O) at the indicated time points. Data information: mean ± s.e.m. of n=5-6 mice (O) (*p<0.05; two-sided t-test or one-way ANOVA followed by Tukey's test).


WT and Batf KO mice were orally infected by gavage with 2x109 cfu / ml of Citrobacter rodentium ( Cr ) expressing GFP Cr infected WT and Batf KO were sacrificed on day 4 and day 10 . Cells were isolated from the colon , re - stimulated with PMA and ionomycin , and stained intracellularly for flow cytometric analysis of cytokine production and Foxp3 expression presented as density plots ( P ) . Data are gated on viable CD4 + TCRβ + . Data information : Data are representative of two independent

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

WT and Batf KO mice were orally infected by gavage with 2x109 cfu/ml of Citrobacter rodentium (Cr) expressing GFP Cr infected WT and Batf KO were sacrificed on day 4 and day 10. Cells were isolated from the colon, re-stimulated with PMA and ionomycin, and stained intracellularly for flow cytometric analysis of cytokine production and Foxp3 expression presented as density plots (P). Data are gated on viable CD4+TCRβ+. Data information: Data are representative of two independent experiments


Naïve WT and Batf KO CD4 + CD62Lhi T cells were cultured under Th17 polarizing conditions for 5 days . ChIP - seq for transcription factor ( Batf ) and histone modifications ( H3K4me3 , H3K27me3 , and H3K27ac ) was performed using day 5 WT and Batf KO Th17 cells ( A Data information : Data are representative of two independent experiments with similar results ( A ,

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

Naïve WT and Batf KO CD4+CD62Lhi T cells were cultured under Th17 polarizing conditions for 5 days. ChIP-seq for transcription factor (Batf) and histone modifications (H3K4me3, H3K27me3, and H3K27ac) was performed using day 5 WT and Batf KO Th17 cells (A Data information: Data are representative of two independent experiments with similar results (A,


J , Naïve WT - 2BiT and Batf KO - 2BiT CD4 + CD62Lhi T cells were cultured under Th17 polarizing conditions . IL - 2 - Thy1 . 1 production comparing WT - 2BiT and Batf KO - 2BiT cells was measured by flow cytometry at the indicated time points and frequencies of positive cells determined . Data information : Data are representative of or mean ± s . e . m . of 3 - 5 independent experiments ( * p < 0 . 05 ; two - sided t - test ) .

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

J, Naïve WT-2BiT and Batf KO-2BiT CD4+CD62Lhi T cells were cultured under Th17 polarizing conditions. IL-2-Thy1.1 production comparing WT-2BiT and Batf KO-2BiT cells was measured by flow cytometry at the indicated time points and frequencies of positive cells determined. Data information: Data are representative of or mean ± s.e.m. of 3-5 independent experiments (*p<0.05; two-sided t-test).


L and M , ELISA was used to quantify IL - 2 production in supernatants derived from cells in Figures 1G ( L ) and 1J ( M ) after 24 h of anti - CD3 stimulation . Data information : Data are representative of or mean ± s . e . m . of 3 - 5 independent experiments L - M ) ( * p < 0 . 05 ; two - sided t - test ) .

L and M, ELISA was used to quantify IL-2 production in supernatants derived from cells in Figures 1G (L) and 1J (M) after 24 h of anti-CD3 stimulation. Data information: Data are representative of or mean ± s.e.m. of 3-5 independent experiments L-M) (*p<0.05; two-sided t-test).


ChIP - seq for Stat3 and Stat5 was performed after 1 h of IL - 6 and IL - 2 stimulation , respectively , using WT and Batf KO cells that were cultured under Th17 polarizing conditions for 5 days . Histograms show Batf , Stat3 , and Stat5 binding occupied around ± 1 kb of peak centers of co - bound Stat3 - Stat5 sites that were annotated near Th1 , Th17 , or Treg - specific genes ( C ) . Data information : Data are mean ± s . e . m . representative of two independent experiments with similar results ( c ( * p < 0 . 05 ; two - sided t - test ) .

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

ChIP-seq for Stat3 and Stat5 was performed after 1 h of IL-6 and IL-2 stimulation, respectively, using WT and Batf KO cells that were cultured under Th17 polarizing conditions for 5 days. Histograms show Batf, Stat3, and Stat5 binding occupied around ±1 kb of peak centers of co-bound Stat3-Stat5 sites that were annotated near Th1, Th17, or Treg-specific genes (C). Data information: Data are mean ± s.e.m. representative of two independent experiments with similar results (c (*p<0.05; two-sided t-test).


D , ChIP - seq for Stat3 and Stat5 was performed after 1 h of IL - 6 and IL - 2 stimulation , respectively , using WT and Batf KO cells that were cultured under Th17 polarizing conditions for 5 days . Tracks show Batf , Stat5 , and Stat3 ChIP - seq data compared between WT and Batf KO Th17 in the indicated gene loci ( WT , grey ; Batf KO , red ) . Red arrows indicate differential Stat5 binding comparing WT and Batf KO cells ( D ) . Data information : Data are mean ± s . e . m . representative of two independent experiments with similar results d ) , or ( * p < 0 . 05 ; two - sided t - test ) .

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

D, ChIP-seq for Stat3 and Stat5 was performed after 1 h of IL-6 and IL-2 stimulation, respectively, using WT and Batf KO cells that were cultured under Th17 polarizing conditions for 5 days. Tracks show Batf, Stat5, and Stat3 ChIP-seq data compared between WT and Batf KO Th17 in the indicated gene loci (WT, grey; Batf KO, red). Red arrows indicate differential Stat5 binding comparing WT and Batf KO cells (D). Data information: Data are mean ± s.e.m. representative of two independent experiments with similar results d), or (*p<0.05; two-sided t-test).


B , Naïve WT and Batf KO CD4 + CD62Lhi T cells were cultured under Th17 polarizing conditions for 5 days . The kinetics of Ets1 and Runx1 gene expression was assessed by RT - PCR in the indicated time points ( data normalized to naïve WT cells ) . Data information : Data are mean ± s . e . m . of 3 independent experiments ( B ) ( * p < 0 . 05 ; two - sided t - test ) . RQ , relative quantification .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, Naïve WT and Batf KO CD4+CD62Lhi T cells were cultured under Th17 polarizing conditions for 5 days. The kinetics of Ets1 and Runx1 gene expression was assessed by RT-PCR in the indicated time points (data normalized to naïve WT cells). Data information: Data are mean ± s.e.m. of 3 independent experiments (B) (*p<0.05; two-sided t-test). RQ, relative quantification.


Tracks show Ets1 and Runx1 ChIP - seq data from WT and Batf KO Th17 - polarized cells at the indicated gene loci ( D ; WT , grey ; Batf KO , red ) . Arrows indicate differential Ets1 and Runx1 binding with ( red ) or without ( blue ) the presence of Batf binding comparing WT and Batf KO cells . Ets1 ( E ) or Runx1 ( F ) peaks from WT Th1 , Treg , or both ( Th1 - Treg ) were integrated with enriched Ets1 or Runx1 peaks in Batf KO compared to WT Th17 cells ( Fig . 4C ) to separate peaks into unique and overlapping clusters .

Tracks show Ets1 and Runx1 ChIP-seq data from WT and Batf KO Th17-polarized cells at the indicated gene loci (D; WT, grey; Batf KO, red). Arrows indicate differential Ets1 and Runx1 binding with (red) or without (blue) the presence of Batf binding comparing WT and Batf KO cells. Ets1 (E) or Runx1 (F) peaks from WT Th1, Treg, or both (Th1-Treg) were integrated with enriched Ets1 or Runx1 peaks in Batf KO compared to WT Th17 cells (Fig. 4C) to separate peaks into unique and overlapping clusters.


Naïve WT and Batf KO CD4 + CD62Lhi T cells cultured under Th17 - polarizing conditions were treated with or without 1 uM Stat5 inhibitor . On day 5 , cells were harvested and used for RNA extraction to assess gene expression ( A ; data were normalized to WT DMSO ; Foxp3 , unstimulated ; Il17a and Ifng , 6 h 2 ug / ml anti - CD3 restimulation ) Data information : Data are mean ± s . e . m . of 4 independent experiments . ( * p < 0 . 05 ; two - sided t - test or one - way ANOVA followed by Tukey ' s test ) . a . u . , arbitrary unit .

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

Naïve WT and Batf KO CD4+CD62Lhi T cells cultured under Th17-polarizing conditions were treated with or without 1 uM Stat5 inhibitor. On day 5, cells were harvested and used for RNA extraction to assess gene expression (A; data were normalized to WT DMSO; Foxp3, unstimulated; Il17a and Ifng, 6 h 2 ug/ml anti-CD3 restimulation) Data information: Data are mean ± s.e.m. of 4 independent experiments. (*p<0.05; two-sided t-test or one-way ANOVA followed by Tukey's test). a.u., arbitrary unit.


Naïve WT and Batf KO CD4 + CD62Lhi T cells cultured under Th17 - polarizing conditions were treated with or without 1 uM Stat5 inhibitor . restimulated with 2 ug / ml anti - CD3 for 24 h to assess cytokine production by ELISA ( B ) . Data information : Data are mean ± s . e . m . of 4 independent experiments . ( * p < 0 . 05 ; two - sided t - test or one - way ANOVA followed by Tukey ' s test ) . a . u . , arbitrary unit .

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

Naïve WT and Batf KO CD4+CD62Lhi T cells cultured under Th17-polarizing conditions were treated with or without 1 uM Stat5 inhibitor. restimulated with 2 ug/ml anti-CD3 for 24 h to assess cytokine production by ELISA (B). Data information: Data are mean ± s.e.m. of 4 independent experiments. (*p<0.05; two-sided t-test or one-way ANOVA followed by Tukey's test). a.u., arbitrary unit.


Naïve WT and Batf KO CD4 + CD62Lhi T cells cultured under Th17 - polarizing conditions were treated with or without 1 uM Stat5 inhibitor . WT and Batf KO Th17 - polarized cells were collected on day 5 of culture and used to performed ChIP using antibodies to Stat5 , Batf , Ets1 , Runx1 , Stat3 and p300 ( C ) . Data information : Data are mean ± s . e . m . of 4 independent experiments . ( * p < 0 . 05 ; two - sided t - test or one - way ANOVA followed by Tukey ' s test ) . a . u . , arbitrary unit .

Naïve WT and Batf KO CD4+CD62Lhi T cells cultured under Th17-polarizing conditions were treated with or without 1 uM Stat5 inhibitor. WT and Batf KO Th17-polarized cells were collected on day 5 of culture and used to performed ChIP using antibodies to Stat5, Batf, Ets1, Runx1, Stat3 and p300 (C). Data information: Data are mean ± s.e.m. of 4 independent experiments. (*p<0.05; two-sided t-test or one-way ANOVA followed by Tukey's test). a.u., arbitrary unit.


Nuclear extracts from WT Th17 cells were incubated with biotinylated oligonucleotides containing Ets1 - Runx1 - Stat5 - specific binding sites at the indicated genomic regions ( wild type or mutant ( ∆ ) as indicated ) . Immunoblots of precipitated proteins ( D )

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

Nuclear extracts from WT Th17 cells were incubated with biotinylated oligonucleotides containing Ets1-Runx1-Stat5-specific binding sites at the indicated genomic regions (wild type or mutant (∆) as indicated). Immunoblots of precipitated proteins (D)


E , Nuclear extracts from WT Th17 cells were incubated with biotinylated oligonucleotides containing Ets1 - Runx1 - Stat5 - specific binding sites at the indicated genomic regions ( wild type or mutant ( ∆ ) as indicated ) . densitometry measurements ( E ) . Data information : Data are mean ± s . e . m . of 4 independent experiments . ( * p < 0 . 05 ; two - sided t - test or one - way ANOVA followed by Tukey ' s test ) . a . u . , arbitrary unit .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, Nuclear extracts from WT Th17 cells were incubated with biotinylated oligonucleotides containing Ets1-Runx1-Stat5-specific binding sites at the indicated genomic regions (wild type or mutant (∆) as indicated). densitometry measurements (E). Data information: Data are mean ± s.e.m. of 4 independent experiments. (*p<0.05; two-sided t-test or one-way ANOVA followed by Tukey's test). a.u., arbitrary unit.


Naïve CD4 + CD62Lhi T cells from IL - 17a reporter mice ( Il17aeGFP ) were cultured under Th17 polarizing conditions . On day 5 , eGFP + / IL - 17a + cells were sorted ( Th17 ( R1 ) ) and cultured for two additional rounds in the presence of anti - CD3 alone ( TCR ( R3 ) ) or Th17 polarizing cytokines ( Th17 ( R3 ) ) . Ets1 , Runx1 , and Stat5 binding at the indicated genomic regions by ChIP - qPCR ( C ) . Data information : Data are mean ± s . e . m . of 3 independent experiments . ( * p < 0 . 05 ; * * p < 0 . 01 ; one - way ANOVA followed by Tukey ' s test ) .

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

Naïve CD4+CD62Lhi T cells from IL-17a reporter mice (Il17aeGFP) were cultured under Th17 polarizing conditions. On day 5, eGFP+/IL-17a+ cells were sorted (Th17(R1)) and cultured for two additional rounds in the presence of anti-CD3 alone (TCR(R3)) or Th17 polarizing cytokines (Th17(R3)). Ets1,Runx1, and Stat5 binding at the indicated genomic regions by ChIP-qPCR (C). Data information: Data are mean ± s.e.m. of 3 independent experiments. (*p<0.05; **p<0.01; one-way ANOVA followed by Tukey's test).


Blue native gel electrophoresis ( BNGdataE ) of mouse ( M ) C57BL / 6J ( 111 ) , CD1 ( 113 ) and zebrafish ( ZF ) skeletal muscle digitonin - solubilized mitochondria . Immunodetection of the indicated proteins after

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

Blue native gel electrophoresis (BNGdataE) of mouse (M) C57BL/6J (111), CD1 (113) and zebrafish (ZF) skeletal muscle digitonin-solubilized mitochondria. Immunodetection of the indicated proteins after BNGE


Blue native gel electrophoresis ( BNGdataE ) of mouse ( M ) C57BL / 6J ( 111 ) , CD1 ( 113 ) and zebrafish ( ZF ) skeletal muscle digitonin - solubilized mitochondria . ( C ) in - gel activity for CI and ( D ) CIV ( shown is a representative gel from two technical and two biological replicates ) .

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

Blue native gel electrophoresis (BNGdataE) of mouse (M) C57BL/6J (111), CD1 (113) and zebrafish (ZF) skeletal muscle digitonin-solubilized mitochondria. (C) in-gel activity for CI and (D) CIV (shown is a representative gel from two technical and two biological replicates).


BNGE of whole - body zebrafish digitonin - solubilized mitochondria of scaf1Δ1 / Δ1 ( - / - ) and its respective scaf1 + / + counterpart . Immunodetection of the indicated

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

BNGE of whole-body zebrafish digitonin-solubilized mitochondria of scaf1Δ1/Δ1 (-/-) and its respective scaf1+/+ counterpart. Immunodetection of the indicated proteins


BNGE of whole - body zebrafish digitonin - solubilized mitochondria of scaf1Δ1 / Δ1 ( - / - ) and its respective scaf1 + / + counterpart . ( G ) in - gel activity of CI and ( H ) CIV ( representative gel from two technical and three biological replicates ) .

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

BNGE of whole-body zebrafish digitonin-solubilized mitochondria of scaf1Δ1/Δ1 (-/-) and its respective scaf1+/+ counterpart. (G) in-gel activity of CI and (H) CIV (representative gel from two technical and three biological replicates).


I , 2D BNGE / SDS electrophoresis : 1st dimension with digitonin ( Dig ) and 2nd dimension with SDS , followed by immunoblotting with the indicated antibodies to identify the proteins detected by the commercial anti - SCAF1 antibody . Asterisks indicate missing bands in scaf1Δ1 / Δ1 .

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

I, 2D BNGE/SDS electrophoresis: 1st dimension with digitonin (Dig) and 2nd dimension with SDS, followed by immunoblotting with the indicated antibodies to identify the proteins detected by the commercial anti-SCAF1 antibody. Asterisks indicate missing bands in scaf1Δ1/Δ1.


A , Quantitative data - independent scanning ( DiS ) mass spectrometry protein profiles for CI , CIII and CIV . Vertical numbers indicate the BNGE gel slices . Left and right profiles correspond to scaf1 + / + and scaf1Δ1 / Δ1 animals , respectively . Red heatmap corresponds to the E - score from two proteotypic Scaf1 - derived tryptic peptides spanning sequences ascribed to the CIII interacting site ( in green ) and to the CIV interacting site ( in yellow ) . Thick blue line , marked with an asterisk , indicates the putative proteolytic site in Scaf1 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, Quantitative data-independent scanning (DiS) mass spectrometry protein profiles for CI, CIII and CIV. Vertical numbers indicate the BNGE gel slices. Left and right profiles correspond to scaf1+/+ and scaf1Δ1/Δ1 animals, respectively. Red heatmap corresponds to the E-score from two proteotypic Scaf1-derived tryptic peptides spanning sequences ascribed to the CIII interacting site (in green) and to the CIV interacting site (in yellow). Thick blue line, marked with an asterisk, indicates the putative proteolytic site in Scaf1.


A , B , Representative images from scaf1 + / + and scaf1 - / - ( A ) female and ( B ) male adult zebrafish .

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

A, B, Representative images from scaf1+/+ and scaf1-/- (A) female and (B) male adult zebrafish.


c - f , Size of scaf1Δ1 / Δ1 and scaf1Δ2 / Δ2 ( scaf1 - / - ) fish in comparison with their respective scaf1 + / + lines , ( C ) length and ( E ) weight of females ( Δ1 + / + n = 10 , Δ1 - / - n = 12 , Δ2 + / + n = 24 , Δ2 - / - n = 18 ) ; ( D ) length and ( F ) weight of males ( Δ1 + / + n = 16 , Δ1 - / - n = 13 , Δ2 + / + n = 13 , Δ2 - / - n = 23 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-f, Size of scaf1Δ1/ Δ1 and scaf1Δ2/ Δ2 (scaf1-/-) fish in comparison with their respective scaf1+/+ lines, (C) length and (E) weight of females (Δ1 +/+ n=10, Δ1 -/- n=12, Δ2 +/+ n=24, Δ2 -/- n=18); (D) length and (F) weight of males (Δ1 +/+ n=16, Δ1 -/- n=13, Δ2 +/+ n=13, Δ2 -/- n=23).


G - K , Adipose tissue measurements on hematoxylin - eosin ( H & E ) - stained adult zebrafish sagittal sections . ( G , I ) Adipose tissue area per total section area ( average of 3 sections / biological replicate ) and ( H , J ) adipocyte size ( average of 20 - 30 adipocytes of ventral adipose tissue per biological replicate ) of females ( G , H ) ( Δ1 n = 8 , Δ2 n = 8 , same number of animals for homozygous mutants and controls ) and males ( I , J ) ( Δ1 n = 8 , Δ2 n = 7 , same number of animals for homozygous mutants and controls ) . K , Representative images of ventral fat deposits in females ( dotted lines ) .

G-K, Adipose tissue measurements on hematoxylin-eosin (H&E)-stained adult zebrafish sagittal sections. (G, I) Adipose tissue area per total section area (average of 3 sections/biological replicate) and (H, J) adipocyte size (average of 20-30 adipocytes of ventral adipose tissue per biological replicate) of females (G, H) (Δ1 n=8, Δ2 n=8, same number of animals for homozygous mutants and controls) and males (I, J)(Δ1 n=8, Δ2 n=7, same number of animals for homozygous mutants and controls). K, Representative images of ventral fat deposits in females (dotted lines).


L - N , Effect of Scaf1 loss of function on female fertility . ( L ) Number of eggs per clutch ( Δ1 + / + n = 12 , Δ1 - / - n = 13 , Δ2 + / + n = 13 , Δ2 - / - n = 10 ) . ( M ) Quantification of mature ovary follicles per ovary section ( average of three sections / biological replicate ) ( Δ1 n = 5 , Δ2 n = 8 ; same number of animals for homozygous scaf1 + / + and scaf1 - / - ) . ( N ) Representative images of H & E - stained ovaries . Dotted lines delineate adipose 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

L-N, Effect of Scaf1 loss of function on female fertility. (L) Number of eggs per clutch (Δ1 +/+ n=12, Δ1 -/- n=13, Δ2 +/+ n=13, Δ2 -/- n=10). (M) Quantification of mature ovary follicles per ovary section (average of three sections/biological replicate) (Δ1 n=5, Δ2 n=8; same number of animals for homozygous scaf1+/+ and scaf1-/-). (N) Representative images of H&E-stained ovaries. Dotted lines delineate adipose tissue.


Transmission electron microscopy image of cardiac muscle from scaf1Δ1 / Δ1 ( n = 3 ) and scaf1 + / + fish ( n = 3 ) . ( A ) Representative images showing mitochondria .

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

Transmission electron microscopy image of cardiac muscle from scaf1Δ1/Δ1 (n=3) and scaf1+/+ fish (n=3). (A) Representative images showing mitochondria.


Transmission electron microscopy image of cardiac muscle from scaf1Δ1 / Δ1 ( n = 3 ) and scaf1 + / + fish ( n = 3 ) . ( B ) Mitochondria size ( 100 - 150 mitochondria per biological sample ) . ( C ) Cristae lumen width ( average of three cristae per mitochondria , 20 mitochondria per biological sample ) . Different biological replicates are represented with different color tones .

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

Transmission electron microscopy image of cardiac muscle from scaf1Δ1/Δ1 (n=3) and scaf1+/+ fish (n=3). (B) Mitochondria size (100-150 mitochondria per biological sample). (C) Cristae lumen width (average of three cristae per mitochondria, 20 mitochondria per biological sample). Different biological replicates are represented with different color tones.


D , E , Mitochondrial DNA copy number per nuclear copy number in muscle in females ( D ) and males ( E ) ( Δ1 n = 6 , Δ2 n = 6 and same number for their respective controls ) .

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

D, E, Mitochondrial DNA copy number per nuclear copy number in muscle in females (D) and males (E) (Δ1 n=6, Δ2 n=6 and same number for their respective controls).


F , Survival curve of 4 days post - fertilization embryos treated with different concentrations of the indicated OXPHOS inhibitors ( three experimental replicates per biological replicate and three biological replicates ) .

O O O O O O O O O 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, Survival curve of 4 days post-fertilization embryos treated with different concentrations of the indicated OXPHOS inhibitors (three experimental replicates per biological replicate and three biological replicates).


G , H , Oxygen consumption of 48 hours post - fertilization embryos using the XFe24 Seahorse analyzer , ( G ) Representative oxygen consumption rate ( OCR ) profile along time and ( H ) maximum OCR ( Δ1 n = 11 , Δ2 n = 11 , n = 10 , n = 11 respectively for their controls ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, H, Oxygen consumption of 48 hours post-fertilization embryos using the XFe24 Seahorse analyzer, (G) Representative oxygen consumption rate (OCR) profile along time and (H) maximum OCR (Δ1 n=11, Δ2 n=11, n=10, n=11 respectively for their controls).


I , Maximum uncoupled ( FCCP ) OCR in isolated mitochondria from adult fish ( male and females Δ1 n = 4 and Δ2 n = 4 , and same number for their respective controls ) with the indicated site I [ pyruvate ( Pyr ) , glutamate ( Glu ) , malate ( Mal ) ] or site II [ succinate ( Succ ) ] substrates .

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

I, Maximum uncoupled (FCCP) OCR in isolated mitochondria from adult fish (male and females Δ1 n=4 and Δ2 n=4, and same number for their respective controls) with the indicated site I [pyruvate (Pyr), glutamate (Glu), malate (Mal)] or site II [succinate (Succ)] substrates.


J , K , Respiratory control ratio ( RCR ) ( State 3 / State4 ) ( J ) and P / O ratio ( K ) in isolated mitochondria from adult fish ( male and females Δ1 n = 4 , and same number for their respective controls ) with the indicated substrates .

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

J, K, Respiratory control ratio (RCR)(State 3/State4) (J) and P/O ratio (K) in isolated mitochondria from adult fish (male and females Δ1 n=4, and same number for their respective controls) with the indicated substrates.


A , Representative images of scaf1 - / - and scaf1 + / + fish fed with the indicated diets .

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A, Representative images of scaf1-/- and scaf1+/+ fish fed with the indicated diets.


B , C , Changes in ( B ) length and ( C ) weight over time ( Δ1 + / + n = 10 , Δ1 - / - n = 10 , Δ2 + / + n = 10 , Δ2 - / - n = 12 - 13 .

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

B, C, Changes in (B) length and (C) weight over time (Δ1 +/+ n=10, Δ1 -/- n=10, Δ2 +/+ n=10, Δ2 -/- n=12-13.


D - F , Adipose tissue measurements on hematoxylin - eosin ( H & E ) - stained adult zebrafish sagittal sections . ( D ) Adipose tissue area per total section area ( average of three sections / biological replicate ) and ( E ) adipocyte size ( average of 20 - 30 adipocytes of ventral adipose tissue per biological replicate ) ( Δ1 + / + n = 3 , Δ2 n = 8 ) . scaf1Δ1 and scaf1Δ2 are represented with circles and squares , respectively .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-F, Adipose tissue measurements on hematoxylin-eosin (H&E)-stained adult zebrafish sagittal sections. (D) Adipose tissue area per total section area (average of three sections/biological replicate) and (E) adipocyte size (average of 20-30 adipocytes of ventral adipose tissue per biological replicate) (Δ1 +/+ n=3, Δ2 n=8). scaf1Δ1 and scaf1Δ2 are represented with circles and squares, respectively.


Adipose tissue measurements on hematoxylin - eosin ( H & E ) - stained adult zebrafish sagittal sections . ( F ) Representative images of ventral fat deposits ( dotted lines ) .

Adipose tissue measurements on hematoxylin-eosin (H&E)-stained adult zebrafish sagittal sections. (F) Representative images of ventral fat deposits (dotted lines).


G , Number of eggs per clutch ( standard diet Δ1 + / + n = 8 , Δ1 - / - n = 8 , Δ2 + / + n = 7 , Δ2 - / - n = 12 , double diet Δ1 + / + n = 7 , Δ1 - / - n = 8 , Δ2 + / + n = 8 , Δ2 - / - n = 9 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, Number of eggs per clutch (standard diet Δ1 +/+ n=8, Δ1 -/- n=8, Δ2 +/+ n=7, Δ2 -/- n=12, double diet Δ1 +/+ n=7, Δ1 -/- n=8, Δ2 +/+ n=8, Δ2 -/- n=9).


H , Representative images of H & E - stained ovaries . Black dotted lines outline the ovaries , blue dotted line indicates a mature follicle . I , Quantification of mature ovary follicles per ovary section ( average of three sections / biological sample ) ( Δ1 n = 2 - 3 , Δ2 n = 6 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, Representative images of H&E-stained ovaries. Black dotted lines outline the ovaries, blue dotted line indicates a mature follicle. I, Quantification of mature ovary follicles per ovary section (average of three sections/biological sample) (Δ1 n=2-3, Δ2 n=6).


A , B , Representative images of scaf1 - / - and scaf1 + / + females ( A ) and males ( B ) fed with the indicated diets .

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

A, B, Representative images of scaf1-/- and scaf1+/+ females (A) and males (B) fed with the indicated diets.


C , D , Length of females ( C ) and males ( D ) after the indicated diets ( Δ1 + / + n = 5 , Δ1 - / - n = 5 , Δ2 + / + n = 5 , Δ2 - / - n = 5 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, D, Length of females (C) and males (D) after the indicated diets (Δ1 +/+ n=5, Δ1 -/- n=5, Δ2 +/+ n=5, Δ2 -/- n=5).


E , F , Weight of females ( E ) and males ( F ) after the indicated diets ( Δ1 + / + n = 5 , Δ1 - / - n = 5 , Δ2 + / + n = 5 , Δ2 - / - n = 5 ) .

E, F, Weight of females (E) and males (F) after the indicated diets (Δ1 +/+ n=5, Δ1 -/- n=5, Δ2 +/+ n=5, Δ2 -/- n=5).


G , number of eggs per clutch ( standard diet : Δ1 + / + n = 4 , Δ1 - / - n = 3 , Δ2 + / + n = 3 , Δ2 - / - n = 2 , double diet : Δ1 + / + n = 5 , Δ1 - / - , n = 5 Δ2 + / + n = 1 , Δ2 - / - n = 2 ) . H , Quantification of mature ovary follicles per ovary section in haematoxylin - eosin ( H & E ) histological sections ( average of three sections / biological sample ) ( Δ1 n = 3 , Δ2 n = 3 ) .

G, number of eggs per clutch (standard diet: Δ1 +/+ n=4, Δ1 -/- n=3, Δ2 +/+ n=3, Δ2 -/- n=2, double diet: Δ1 +/+ n=5, Δ1 -/-, n=5 Δ2 +/+ n=1, Δ2 -/- n=2). H, Quantification of mature ovary follicles per ovary section in haematoxylin-eosin (H&E) histological sections (average of three sections/biological sample) (Δ1 n=3, Δ2 n=3).


adipose tissue quantification in H & E sections ( Δ1 n = 3 , Δ2 n = 3 ) in females and males : adipose tissue area ( I , J )

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

adipose tissue quantification in H&E sections (Δ1 n=3, Δ2 n=3) in females and males : adipose tissue area (I, J)


adipose tissue quantification in H & E sections ( Δ1 n = 3 , Δ2 n = 3 ) in females and males adipocyte size ( K , L ) .

adipose tissue quantification in H&E sections (Δ1 n=3, Δ2 n=3) in females and males adipocyte size (K, L).


A , Immunoblot of the indicated proteins of BNGE from female scaf1 + / + and scaf1 - / - whole zebrafish mitochondria for the indicated diet ( representative of n = 2 ) .

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

A, Immunoblot of the indicated proteins of BNGE from female scaf1+/+ and scaf1-/- whole zebrafish mitochondria for the indicated diet (representative of n=2).


B , Maximum uncoupled ( FCCP ) oxygen consumption rate in whole zebrafish mitochondria ( females Δ1 n = 4 and Δ2 n = 4 , and same number for their respective controls ) with glutamate ( Glu ) , malate ( Mal ) and succinate ( Succ ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O 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, Maximum uncoupled (FCCP) oxygen consumption rate in whole zebrafish mitochondria (females Δ1 n=4 and Δ2 n=4, and same number for their respective controls) with glutamate (Glu), malate (Mal) and succinate (Succ).


RNAseq data from scaf1 + / + and scaf1 - / - skeletal muscle for the indicated diet ( standard diet scaf1 + / + and scaf1 - / - n = 4 , double diet scaf1 + / + n = 3 , scaf1 - / - n = 4 ) . ( C - F ) Volcano plots of differentially expressed genes ( DEGs ) . ( C ) Comparison between scaf1 - / - and scaf1 + / + zebrafish in standard diet . ( D ) Comparison between scaf1 - / - and scaf1 + / + zebrafish in double diet . ( E ) Comparison of scaf1 + / + zebrafish in double diet and standard diet . ( F ) Comparison of scaf1 - / - zebrafish in double diet and standard diet . In blue , significant DEGs ( p - adj < 0 . 05 , log2FC > | 1 | ) , in grey not significant DEGs , red circles represent non - significant differentially regulated OXPHOS genes , green circles represent significant differentially regulated OXPHOS genes , purple scaf1 ( cox7a2l ) .

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

RNAseq data from scaf1+/+ and scaf1-/- skeletal muscle for the indicated diet (standard diet scaf1+/+ and scaf1-/- n=4, double diet scaf1+/+ n=3, scaf1-/- n=4). (C-F) Volcano plots of differentially expressed genes (DEGs). (C) Comparison between scaf1-/- and scaf1+/+ zebrafish in standard diet. (D) Comparison between scaf1-/- and scaf1+/+ zebrafish in double diet. (E) Comparison of scaf1+/+ zebrafish in double diet and standard diet. (F) Comparison of scaf1-/- zebrafish in double diet and standard diet. In blue, significant DEGs (p-adj < 0.05, log2FC > |1|), in grey not significant DEGs, red circles represent non-significant differentially regulated OXPHOS genes, green circles represent significant differentially regulated OXPHOS genes, purple scaf1 (cox7a2l).


B . Detection of sFn14 in conditioned media . HEK293E cells were transfected with either empty vector or a plasmid encoding human Fn14 that bears an N - terminal HA - tag and a C - terminal double FLAG - tag . Conditioned media and lysates of the transfected cells were collected and analyzed by immunoblotting with the indicated antibodies . Calnexin served as a loading control . Shown are representative blots from N = 3 experiments .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Detection of sFn14 in conditioned media. HEK293E cells were transfected with either empty vector or a plasmid encoding human Fn14 that bears an N-terminal HA-tag and a C-terminal double FLAG-tag. Conditioned media and lysates of the transfected cells were collected and analyzed by immunoblotting with the indicated antibodies. Calnexin served as a loading control. Shown are representative blots from N=3 experiments.


C . Generation of sFn14 is sensitive to the γ - secretase inhibitor DAPT . HEK293E cells were transfected with either Fn14 or C99 ( C - terminal fragment of APP ) , both containing a N - terminal HA - tag and C - terminal double FLAG - tag . One day after transfection , cells were treated with γ - secretase inhibitor DAPT ( 1 μM ) , broad - spectrum metalloprotease inhibitor TAPI - 1 ( 50 μM ) , or the corresponding amount of vehicle DMSO as indicated . The conditioned media and the lysates were blotted with anti - HA antibody . Shown are representative blots from N = 4 experiments .

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

C. Generation of sFn14 is sensitive to the γ-secretase inhibitor DAPT. HEK293E cells were transfected with either Fn14 or C99 (C-terminal fragment of APP), both containing a N-terminal HA-tag and C-terminal double FLAG-tag. One day after transfection, cells were treated with γ-secretase inhibitor DAPT (1 μM), broad-spectrum metalloprotease inhibitor TAPI-1 (50 μM), or the corresponding amount of vehicle DMSO as indicated. The conditioned media and the lysates were blotted with anti-HA antibody. Shown are representative blots from N=4 experiments.


E . In vitro γ - secretase cleavage assay . HEK293E cells were transfected with epitope - tagged Fn14 or C99 . Cellular membranes were collected and incubated under indicated conditions for the γ - secretase activity assay . Reactions were terminated and ultracentrifuged . Supernatant ( containing γ - secretase cleavage products ) was used for detecting the ICD fragment while the pellet was used to blot for full length proteins Fn14 or C99 . Blotting is done by anti - Flag antibody . Shown are representative blots from N = 3 experiments .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. In vitro γ-secretase cleavage assay. HEK293E cells were transfected with epitope-tagged Fn14 or C99. Cellular membranes were collected and incubated under indicated conditions for the γ-secretase activity assay. Reactions were terminated and ultracentrifuged. Supernatant (containing γ-secretase cleavage products) was used for detecting the ICD fragment while the pellet was used to blot for full length proteins Fn14 or C99. Blotting is done by anti-Flag antibody. Shown are representative blots from N=3 experiments.


F . sFn14 production requires the proteolytic presenilin subunit of γ - secretase . HEK293 cells stably transfected with APP carrying the Swedish double mutation ( K595N / M596L ) and with a CRISPR / CAS9 - mediated knockout of presenilin 1 ( PS1 KO ) or of both PS1 and PS2 ( PS1 / 2 dKO ) , were transiently transfected with epitope - tagged Fn14 . Conditioned media and lysates of the transfected cells were blotted with the indicated antibodies . C83 is a small C - terminal fragment of APP ( containing the C - terminal 83 amino acids ) , which is also subjected to γ - secretase processing . Upon longer exposure , the C99 ( generated by BACE1 from APP ) is also visible in the PS1 / 2 dKO condition . Shown are representative blots from N = 4 experiments . G . Quantification of results from panel F . sFn14 levels in the conditioned media quantified and normalized to WT sFn14 signal . Data Information : Panel ( G ) shows mean ± SEM , along with p - values calculated by ordinary one - way ANOVA test with Tukey ' s multiple comparison test .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. sFn14 production requires the proteolytic presenilin subunit of γ-secretase. HEK293 cells stably transfected with APP carrying the Swedish double mutation (K595N/M596L) and with a CRISPR/CAS9-mediated knockout of presenilin 1 (PS1 KO) or of both PS1 and PS2 (PS1/2 dKO), were transiently transfected with epitope-tagged Fn14. Conditioned media and lysates of the transfected cells were blotted with the indicated antibodies. C83 is a small C-terminal fragment of APP (containing the C-terminal 83 amino acids), which is also subjected to γ-secretase processing. Upon longer exposure, the C99 (generated by BACE1 from APP) is also visible in the PS1/2 dKO condition. Shown are representative blots from N=4 experiments. G. Quantification of results from panel F. sFn14 levels in the conditioned media quantified and normalized to WT sFn14 signal. Data Information: Panel (G) shows mean ± SEM, along with p-values calculated by ordinary one-way ANOVA test with Tukey's multiple comparison test.


A . Human breast cancer cell line MDA - MB - 231 shows cellular accumulation of Fn14 upon γ - secretase inhibition . The cells were treated overnight with γ - secretase inhibitor DAPT ( 1 μM ) , broad spectrum metalloprotease inhibitor TAPI - 1 ( 50 μM ) , or the corresponding amount of vehicle DMSO as indicated . Lysates were blotted for Fn14 with an antibody that targets the C - terminal end of the protein , or against calnexin as loading control . The asterisk labels an N - terminally truncated form of Fn14 . B . Quantification of blots from panel A . The control condition , where the cells were only treated with vehicle ( DMSO ) , was used as baseline , and its average normalized to 1 . Data Information : All quantification data is shown as mean ± SEM . All the panels have N = 3 biological replicates . For panels B the tested conditions were compared against control ( DMSO ) condition by ordinary one - way ANOVA and Dunnett ' s multiple comparison test . The p - values that are above 0 . 05 have not been included into the panels .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Human breast cancer cell line MDA-MB-231 shows cellular accumulation of Fn14 upon γ-secretase inhibition. The cells were treated overnight with γ-secretase inhibitor DAPT (1 μM), broad spectrum metalloprotease inhibitor TAPI-1 (50 μM), or the corresponding amount of vehicle DMSO as indicated. Lysates were blotted for Fn14 with an antibody that targets the C-terminal end of the protein, or against calnexin as loading control. The asterisk labels an N-terminally truncated form of Fn14. B. Quantification of blots from panel A. The control condition, where the cells were only treated with vehicle (DMSO), was used as baseline, and its average normalized to 1. Data Information: All quantification data is shown as mean ± SEM. All the panels have N=3 biological replicates. For panels B the tested conditions were compared against control (DMSO) condition by ordinary one-way ANOVA and Dunnett's multiple comparison test. The p-values that are above 0.05 have not been included into the panels.


C . Human ovarian cancer cell line SKOV - 3 shows cellular accumulation of Fn14 upon γ - secretase inhibition . The cells were treated overnight with γ - secretase inhibitor DAPT ( 1 μM ) , broad spectrum metalloprotease inhibitor TAPI - 1 ( 50 μM ) , or corresponding amount of vehicle DMSO as indicated . Lysates were blotted for Fn14 with an antibody that targets the C - terminal end of the protein , or against calnexin as loading control . The asterisk labels an N - terminally truncated form of Fn14 . D . Quantification of blot from panel C . The control condition , where the cells were only treated with vehicle ( DMSO ) , was used as baseline , and its average normalized to 1 . Data Information : All quantification data is shown as mean ± SEM . All the panels have N = 3 biological replicates . For panels D , the tested conditions were compared against control ( DMSO ) condition by ordinary one - way ANOVA and Dunnett ' s multiple comparison test . The p - values that are above 0 . 05 have not been included into the panels .

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C. Human ovarian cancer cell line SKOV-3 shows cellular accumulation of Fn14 upon γ-secretase inhibition. The cells were treated overnight with γ-secretase inhibitor DAPT (1 μM), broad spectrum metalloprotease inhibitor TAPI-1 (50 μM), or corresponding amount of vehicle DMSO as indicated. Lysates were blotted for Fn14 with an antibody that targets the C-terminal end of the protein, or against calnexin as loading control. The asterisk labels an N-terminally truncated form of Fn14. D. Quantification of blot from panel C. The control condition, where the cells were only treated with vehicle (DMSO), was used as baseline, and its average normalized to 1. Data Information: All quantification data is shown as mean ± SEM. All the panels have N=3 biological replicates. For panels D, the tested conditions were compared against control (DMSO) condition by ordinary one-way ANOVA and Dunnett's multiple comparison test. The p-values that are above 0.05 have not been included into the panels.


A . MDA - MB - 231 cells were transfected with an siRNA pool against human Fn14 or non - targeting control ( Ctrl ) siRNA . A day after transfection , the cells were treated with γ - secretase inhibitor DAPT ( 1 μM ) or vehicle overnight . The lysate was collected and blotted against Fn14 ( C - terminal antibody ) or calnexin as loading control . Shown are representative blots from N = 4 experiments . B . Quantification of blot in panel A . The control condition where the cells were only treated with vehicle DMSO and non - targeting siRNA ( DMSO + siCtrl ) was used as baseline , and its average normalized to 1 . N = 4 experiments . Data information : All quantification data is shown as mean ± SEM . The tested conditions were compared against control ( DMSO + siCtrl ) condition by ordinary one - way ANOVA and Dunnett ' s multiple comparison test . The p - values that are above 0 . 05 have not been included into the panels .

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A. MDA-MB-231 cells were transfected with an siRNA pool against human Fn14 or non-targeting control (Ctrl) siRNA. A day after transfection, the cells were treated with γ-secretase inhibitor DAPT (1 μM) or vehicle overnight. The lysate was collected and blotted against Fn14 (C-terminal antibody) or calnexin as loading control. Shown are representative blots from N=4 experiments. B. Quantification of blot in panel A. The control condition where the cells were only treated with vehicle DMSO and non-targeting siRNA (DMSO + siCtrl) was used as baseline, and its average normalized to 1. N=4 experiments. Data information: All quantification data is shown as mean ± SEM. The tested conditions were compared against control (DMSO + siCtrl) condition by ordinary one-way ANOVA and Dunnett's multiple comparison test. The p-values that are above 0.05 have not been included into the panels.


A . Cellular Fn14 in ex vivo glioblastoma samples increased upon γ - secretase inhibition . Primary cells from four different glioblastomas were treated with DAPT ( 1 μM ) or vehicle overnight . Lysates were blotted against Fn14 and calnexin as loading control . B . Quantification of the blot in panel A . For each glioblastoma , the relative ( rel . ) mean intensity of the normalized Fn14 vehicle condition was used for normalization . Data information : All quantification data is shown as mean ± SEM . The tested conditions were compared against their corresponding control ( DMSO ) condition using two - tailed unpaired t - tests . The p - values that are above 0 . 05 have not been included into the panels . For all the panels , three biological replicates were performed .

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A. Cellular Fn14 in ex vivo glioblastoma samples increased upon γ-secretase inhibition. Primary cells from four different glioblastomas were treated with DAPT (1 μM) or vehicle overnight. Lysates were blotted against Fn14 and calnexin as loading control. B. Quantification of the blot in panel A. For each glioblastoma, the relative (rel.) mean intensity of the normalized Fn14 vehicle condition was used for normalization. Data information: All quantification data is shown as mean ± SEM. The tested conditions were compared against their corresponding control (DMSO) condition using two-tailed unpaired t-tests. The p-values that are above 0.05 have not been included into the panels. For all the panels, three biological replicates were performed.


B . Plasma samples from 10 controls and GBM patients with an initial diagnosis of GBM ( iGBM , 40 patients ) or a recurrent GBM ( rGBM , 20 patients ) were collected and sFn14 levels were measured by ELISA . Data information : For panel B median of the measurements , along with interquartile range borders are displayed . In panel B , ordinary one - way ANOVA with Tukey ' s multiple comparison test was applied , but no value passed our p - value display threshold of 0 . 05 .

B. Plasma samples from 10 controls and GBM patients with an initial diagnosis of GBM (iGBM, 40 patients) or a recurrent GBM (rGBM, 20 patients) were collected and sFn14 levels were measured by ELISA. Data information: For panel B median of the measurements, along with interquartile range borders are displayed. In panel B, ordinary one-way ANOVA with Tukey's multiple comparison test was applied, but no value passed our p-value display threshold of 0.05.


C . Plasma samples from 10 ovarian cancer patients with low / middle and 19 patients with high tumor burden were collected and sFn14 levels were measured by ELISA . The tumor burden was clinically determined by computer tomographic scans . Data information : For panel C , median of the measurements , along with interquartile range borders are displayed . For panel C , a two - tailed unpaired t - test was applied , but no value passed our p - value display threshold of 0 . 05 .

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C. Plasma samples from 10 ovarian cancer patients with low/middle and 19 patients with high tumor burden were collected and sFn14 levels were measured by ELISA. The tumor burden was clinically determined by computer tomographic scans. Data information: For panel C, median of the measurements, along with interquartile range borders are displayed. For panel C, a two-tailed unpaired t-test was applied, but no value passed our p-value display threshold of 0.05.


D . Serum samples from 10 patients with refractory multiple myeloma were collected pre - and post - treatment with 25 mg LY3039478 administered in 3 daily doses over 5 days . sFn14 levels in these samples are displayed ( N = 10 ) . Data information : For panel D , ratio - paired t - test was used to compare the sFn14 levels pre - and post - treatment .

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D. Serum samples from 10 patients with refractory multiple myeloma were collected pre- and post-treatment with 25 mg LY3039478 administered in 3 daily doses over 5 days. sFn14 levels in these samples are displayed (N=10). Data information: For panel D, ratio-paired t-test was used to compare the sFn14 levels pre- and post-treatment.


A Representative IF image of NPCs stained for Nestin , FoxG1 and Pank2 .

A Representative IF image of NPCs stained for Nestin, FoxG1 and Pank2.


B NPCs differentiated into neurons by overexpressing Ngn2 ( one representative experiment is shown ) . Two weeks after the infection differentiated NPC were positive for neuronal markers βIII - tubulin ( Tuj1 ) , Map2 , NeuN and humannuclei ( hNu ) and synaptic markers , the voltage - gated Na + channels ( PanNav ) and the vesicular glutamate transporter 1 ( VGlut1 ) .

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B NPCs differentiated into neurons by overexpressing Ngn2 (one representative experiment is shown). Two weeks after the infection differentiated NPC were positive for neuronal markers βIII-tubulin (Tuj1), Map2, NeuN and humannuclei (hNu) and synaptic markers, the voltage-gated Na+ channels (PanNav) and the vesicular glutamate transporter 1 (VGlut1).


C Western blot of soluble cell homogenates from humanneurons probed with PANK2 and β - actin antibodies ( arrows ) . Asterisk indicates nonspecific band . Data are representative of three independent experiments .

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C Western blot of soluble cell homogenates from humanneurons probed with PANK2 and β-actin antibodies (arrows). Asterisk indicates nonspecific band. Data are representative of three independent experiments.


D Plots showing the total dendritic length and branching points . Data presented as mean and + SEM from at least three independent experiments . A total of 38 neurons were counted for each sample . Statistics were determined by the t - test and resulted not significant .

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D Plots showing the total dendritic length and branching points. Data presented as mean and + SEM from at least three independent experiments. A total of 38 neurons were counted for each sample. Statistics were determined by the t-test and resulted not significant.


E Representative example of co - culture containing control ( green ) , PKAN ( red ) humanneurons , and E18 cortical miceneurons . Control and PKAN NPCs were infected with GFP - LV and tdT - LV expressing vectors , respectively , and differentiated for 8 weeks . Scale bars 20 μm .

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E Representative example of co-culture containing control (green), PKAN (red) humanneurons, and E18 cortical miceneurons. Control and PKAN NPCs were infected with GFP-LV and tdT-LV expressing vectors, respectively, and differentiated for 8 weeks. Scale bars 20 μm.


F Examples of electrophysiological properties of humanneurons : control individual ( top ) ; PKAN patient ( bottom ) . Traces on the left represent trains of action potentials induced by injection of a suprathreshold current step through the patch electrode in current - clamp mode . Middle traces show Na + and K + currents ( down - and upward - deflecting from baseline , respectively ) in response to a 60 mV step from a holding voltage of - 70 mV in voltage - clamp mode . Insets on the right display enlarged portions of the traces to magnify fast Na + currents . G Histogram with percentages of recorded cells showing repetitively firing ( z - test ) .

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F Examples of electrophysiological properties of humanneurons: control individual (top); PKAN patient (bottom). Traces on the left represent trains of action potentials induced by injection of a suprathreshold current step through the patch electrode in current-clamp mode. Middle traces show Na+ and K+ currents (down- and upward-deflecting from baseline, respectively) in response to a 60 mV step from a holding voltage of -70 mV in voltage-clamp mode. Insets on the right display enlarged portions of the traces to magnify fast Na+ currents. G Histogram with percentages of recorded cells showing repetitively firing (z-test).


A Representative images of humanneurons cells stained with the mitochondrial membrane potential sensible fluorescent probe TMRM , the neuronal specific anti - NCAM antibody and the nuclear staining Hoechst . Left panel , basal conditions . Right panel , after addition of the mitochondrial uncoupler FCCP . Scale bar 20 μm . Plot showing the quantification of TMRM fluorescence signal from NCAM + neurons . Data presented as means + SEM of three independent experiments ( unpaired , two - tailed t - test ) .

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A Representative images of humanneurons cells stained with the mitochondrial membrane potential sensible fluorescent probe TMRM, the neuronal specific anti-NCAM antibody and the nuclear staining Hoechst. Left panel, basal conditions. Right panel, after addition of the mitochondrial uncoupler FCCP. Scale bar 20 μm. Plot showing the quantification of TMRM fluorescence signal from NCAM+neurons. Data presented as means + SEM of three independent experiments (unpaired, two-tailed t-test).


B Representative images of ultrastructural analysis of fixed neurons examined with electron microscope . PANK2 panel represents PKAN neuronal cells overexpressing PANK2 protein . Scale bar 500nm . Mitochondrial size was measured at level of the larger diameter along the perpendicular axis for all the mitochondria in > 30 field ( 200 mitochondria in total ) for each sample ( one - way ANOVA ) .

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B Representative images of ultrastructural analysis of fixed neurons examined with electron microscope. PANK2 panel represents PKAN neuronal cells overexpressing PANK2 protein. Scale bar 500nm. Mitochondrial size was measured at level of the larger diameter along the perpendicular axis for all the mitochondria in >30 field (200 mitochondria in total) for each sample (one-way ANOVA).


C OCR measurements of controls and each PKAN patients analyzed individually and D data obtained by individual analysis and plotted as grouped controls , PKAN patients and PKAN patients overexpressing PANK2 . The plots show OCR normalization to cell number . OCR was measured in basal conditions , and after oligomycin and FCCP addition . Bars indicate means + SEM of three independent experiments ( Two - way ANOVA ) .

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C OCR measurements of controls and each PKAN patients analyzed individually and D data obtained by individual analysis and plotted as grouped controls, PKAN patients and PKAN patients overexpressing PANK2. The plots show OCR normalization to cell number. OCR was measured in basal conditions, and after oligomycin and FCCP addition. Bars indicate means + SEM of three independent experiments (Two-way ANOVA).


A Representative images of neurons stained with the neuron specific anti - NCAM antibody , ROS sensible fluorescent probe DCF , and the nuclei dye Hoechst . Scale bar 20 μm . Plots of the DCF fluorescence signal from NCAM positive controls - and PKAN humanneurons , infected or not with Ngn2 - PANK2 - LV . Data presented as means + SEM of at least three independent experiments ( One - way ANOVA ) .

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A Representative images of neurons stained with the neuron specific anti-NCAM antibody, ROS sensible fluorescent probe DCF, and the nuclei dye Hoechst. Scale bar 20 μm. Plots of the DCF fluorescence signal from NCAM positive controls- and PKAN humanneurons, infected or not with Ngn2-PANK2-LV. Data presented as means + SEM of at least three independent experiments (One-way ANOVA).


B Representative images of neurons stained with ThiolTracker Violet and the anti Tuj1 . Scale bar 20 μm . ThiolTracker Violet fluorescence signal from Tuj1 positive neurons were quantified and shown in the plots . Data presented as means + SEM of at least three independent experiments ( unpaired , two - tailed t - test ) .

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B Representative images of neurons stained with ThiolTracker Violet and the anti Tuj1. Scale bar 20 μm. ThiolTracker Violet fluorescence signal from Tuj1 positive neurons were quantified and shown in the plots. Data presented as means + SEM of at least three independent experiments (unpaired, two-tailed t-test).


A Upper panel : representative images of in - gel enzymatic activity of mitochondrial and cytosolic aconitase ( mAco and cAco , respectively ) . The lower part of the gel was cut , stained with Coomassie blue , and a protein band was used as loading control ( Loading ) . Lower panel : quantification of mAco and cAco enzymatic activity by densitometry .

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A Upper panel: representative images of in-gel enzymatic activity of mitochondrial and cytosolic aconitase (mAco and cAco, respectively). The lower part of the gel was cut, stained with Coomassie blue, and a protein band was used as loading control (Loading). Lower panel: quantification of mAco and cAco enzymatic activity by densitometry.


B Upper panel : Western blot analysis of mitochondrial and cytosolic aconitases . Lower panel : quantification of mAco and cAco by densitometry .

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B Upper panel: Western blot analysis of mitochondrial and cytosolic aconitases. Lower panel: quantification of mAco and cAco by densitometry.


C Hemequantification by absorbance at 400 nm of the soluble cell lysates .

C Hemequantification by absorbance at 400 nm of the soluble cell lysates.


D Upper panel : western blot analysis of transferrin receptor ( TfR1 ) and ferritin ( FtH ) . Lower panel : quantification of TfR1 or FtH normalized on actin by densitometry . All the data are presented as means + SEM of at least three independent experiments ( unpaired , two - tailed t - test ) .

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D Upper panel: western blot analysis of transferrin receptor (TfR1) and ferritin (FtH). Lower panel: quantification of TfR1 or FtH normalized on actin by densitometry. All the data are presented as means + SEM of at least three independent experiments (unpaired, two-tailed t-test).


A Representative images of co - cultures of control and PKAN NPCs infected with GFP - LV and tdT - LV expressing vectors , respectively , at the beginning ( Day 1 ) , after 120 ( Day 120 ) and 150 days of differentiation ( Day 150 ) . Scale bar 20 μm . Plots show the number of green and red humanneurons counted at different time points . All data are presented as means + SEM of at least three independent experiments ( One - way ANOVA ) .

A Representative images of co-cultures of control and PKAN NPCs infected with GFP-LV and tdT-LV expressing vectors, respectively, at the beginning (Day 1), after 120 (Day 120) and 150 days of differentiation (Day 150). Scale bar 20 μm. Plots show the number of green and red humanneurons counted at different time points. All data are presented as means + SEM of at least three independent experiments (One-way ANOVA).


B Example of electrophysiological properties of cultured PKAN humanneurons with or without CoA incubation for 30 days . Repetitive firing activity ( left ) and relatively large Na + and K + currents ( right ) , were restored by CoA . The histogram on the right shows fractions of repetitively firing cells recorded in untreated vs . CoA - treated neurons from control and PKAN patients ( z - test ) .

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B Example of electrophysiological properties of cultured PKAN humanneurons with or without CoA incubation for 30 days. Repetitive firing activity (left) and relatively large Na+ and K+ currents (right), were restored by CoA. The histogram on the right shows fractions of repetitively firing cells recorded in untreated vs. CoA-treated neurons from control and PKAN patients (z-test).