EMBO/SourceData huggingface dataset:
Figure legend: "B. WT, and two independent clones each of 53bp1−/−, Shld2−/−/−, and Shld3−/− CH12 clones were stimulated with CIT for 3 days. The IgM+, IgA+, and Iglo populations were sorted and reanalyzed for expression of IgM and IgA 5 days post sort as well as 12 days post sort (see Figure EV4B). Shown on bar graphs are sorted IgM+, IgA+, and Iglo populations (each column, 1 technical replicate) from WT and mutant CH12 clones, and the percent of cells expressing IgM, IgA, or low for both isotypes (Iglo) after 5 days of culture post-sort. A representative flow plot of 53bp1−/− CH12 3 days post CIT treatment is shown."
NCBI gene IDs:
"Shld2: 75698
Shld3: 113002583
53bp1: 27223"
Figure legend: "A. mRNA expression analysis of IgM and IgA was carried out by RT-PCR for Iglo subclones from WT and two independent 53bp1−/− CH12 (TA and T1) clones. The Iglo subclones derived from the 53bp1−/− CH12 TA clone are listed as D01-D18, while the Iglo subclones derived from the 53bp1−/− CH12 T1 clone are listed as F03-F24. Gels show the RT-PCR analysis for IgM cDNA (top), IgA cDNA (middle), and AID cDNA (bottom) as control."
NCBI gene IDs:
"AID: 
IgA: 238447
IgM: 16019
53bp1: 27223"
Figure legend: "B. mRNA expression analysis as in A of Iglo subclones of two independent Shld2-/-/- (F2 and F3) clones, and IgA+ subclones from Shld2-/-/- clone F3. The Iglo subclones derived from the Shld2-/-/- CH12 F2 clone are listed as H03-H40, while the Iglo subclones derived from the Shld2-/-/-CH12 F3 clone are listed as J02-J109."
NCBI gene IDs:
"Shld2: 75698"
Figure legend: "Long-range PCR of Iglo subclones from WT and two independent 53bp1-/- CH12 (TA and T1) clones. The Iglo subclones derived from the 53bp1−/− CH12 TA clone are listed as D01-D18, while the Iglo subclones derived from the 53bp1−/− CH12 T1 clone are listed as F03-F24. Gels show long-range amplicons (top) and AID genomic DNA PCR control (bottom)."
NCBI gene IDs:
"AID: 
53bp1: 27223"
Figure legend: "Long-range PCR as in B of Iglo subclones of two independent Shld2-/-/- (F2 and F3) clones (left 2 panels), and IgA+ subclones from Shld2-/-/- clone F3."
NCBI gene IDs:
"Shld2: 75698"
Figure legend: " C. HEK293 cells were treated for 8 h with 100 μM H2O2 in the presence or absence of the Nrf2 inhibitor trigonelline (Trig). Heme oxygenase 1 (HMOX-1), glutamate:cysteine ligase (GCL) and GSNOR expressions were evaluated RT-qPCR analyses Results shown are the means ± SEM of n = 3 experiments run in triplicate. *p&lt;0.05; n.s., not significant. "
NCBI gene IDs:
"GSNOR: 128
GCL: 2729
glutamate:cysteine ligase: 2729
Heme oxygenase 1: 3162
HMOX-1: 3162"
Figure legend: " D. RT-qPCR analyses of GSNOR mRNA after 2-to-24 h incubation with 100 μM H2O2. Results shown are the means ± SEM of n = 3 experiments run in triplicate, analyzed using ANOVA with Dunnett multiple comparisons test. n.s., not significant. "
NCBI gene IDs:
"GSNOR: 128"
Figure legend: " I. RT-qPCR analyses of GSNOR mRNA in input and heavy polysome fraction pooled together in H2O2-treated samples (for either 8 or 24 h). H3A mRNA expression level was used as housekeeping gene control. Results represent the means ± SD of n ≥ 6 independent experiments shown as % normalized by H3A. ***, p&lt;0.001 Vehicle: PBS. "
NCBI gene IDs:
"GSNOR: 128
H3A: 8350"
Figure legend: " D. HEK293 cells were transfected with a pool of siRNA against ATM (siATM) or with control siRNA (siScr) for 18 h and treated for additional 24 h with 100 μM H2O2. Basal and phospho-active forms of ATM, together with GSNOR were assessed by Western blot. Vinculin and GAPDH were used as loading controls. Densitometry of GSNOR immunoreactive bands is normalized to GAPDH and expressed as arbitrary units. Values shown represent the means ± SD of n = 3 independent experiments. *, p&lt;0.05; **, p&lt;0.01. "
NCBI gene IDs:
"ATM: 472"
Figure legend: " E. HEK293 cells were transfected with a pool of siRNA against CHK2 (siCHK2) or with control siRNA (siScr) for 18 h and treated for additional 24 h with 100 μM H2O2. Basal and phospho-active forms of CHK2, together with GSNOR were assessed by Western blot. GAPDH was used as loading control. Densitometry of GSNOR immunoreactive bands is normalized to GAPDH and expressed as arbitrary units. Values shown represent the means ± SD of n = 3 independent experiments. *, p&lt;0.05. "
NCBI gene IDs:
"CHK2: 11200"
Figure legend: " G. RT-qPCR analyses of GSNOR mRNA in input and heavy polysome fraction pooled together in H2O2 treated samples in the absence or in the presence of the ATM inhibitor KU55933 (KU). H3A mRNA expression level was used as housekeeping gene control. Results are the means ± SEM of n = 3 independent experiments run in duplicate and shown as %, normalized by H3A. *, p&lt;0.05. "
NCBI gene IDs:
"GSNOR: 128
H3A: 8350"
Figure legend: " I. HEK293 cells were transfected with a pool of siRNA against CHK1 (siCHK1) or with control siRNA (siScr) for 18 h and treated for additional 24 h with 100 μM H2O2. CHK1 and GSNOR were assessed by Western blot analysis of. Densitometry of GSNOR immunoreactive bands is normalized to Vinculin, selected as loading control, and expressed as arbitrary units. Values shown represent the means ± SD of n = 3 independent experiments. *, p&lt;0.05. "
NCBI gene IDs:
"CHK1: 1111"
Figure legend: " F. HEK293 cells were transfected with plasmids coding for the wild-type (WT), C2991L redox mutant (CL) of ATM, or with an empty vector (Empty) (used as negative control) for 40 h and treated for additional 8 h with 100 μM H2O2. Basal and phospho-active forms of ATM, and GSNOR were assessed by Western blot analysis. Vinculin and GAPDH were used as loading controls. Two different exposures (low and high) were selected to highlight differences in P-ATM levels in different experimental settings. "
NCBI gene IDs:
"ATM: 472"
Figure legend: " B. HEK293 cells were transfected with a pool of siRNA against p53 (sip53) or with control siRNA (siScr) for 24 h and treated for additional 24 h with 100 μM H2O2. Basal and phospho-active forms of ATM and CHK2, as well as p53 and GSNOR were assessed by Western blot. Phospho:basal level ratios of ATM and CHK2 (normalized to Vinculin), along with densitometry of GSNOR immunoreactive bands (normalized to GAPDH) are expressed as arbitrary units. Values shown represent the means ± SD of n = 3 independent experiments. *, p&lt;0.05; **, p&lt;0.01; n.s., not significant. "
NCBI gene IDs:
"p53: 7157"
Figure legend: " C. HCT116 cells expressing the wild type form of p53 (p53wt), or an empty vector (Empty, selected as negative control), were treated for 24 h with 100 μM H2O2. Basal and phospho-active forms of ATM and CHK2, p53 and GSNOR were assessed by Western blot. Vinculin and GAPDH were used as loading controls. Phospho:basal level ratios of ATM and CHK2, along with densitometry of p53 and GSNOR immunoreactive bands are expressed as arbitrary units. Values shown represent the means ± SD of n = 3 independent experiments. *, p&lt;0.05; **, p&lt;0.01; n.s., not significant. "
NCBI gene IDs:
"p53: 7157"
Figure legend: " C. HEK293 were transfected for 48 h with siRNA against ATM (siATM), GSNOR (siGSNOR), or control siRNA (scramble, siScr). Afterwards, they were treated for 8 h with 100 μM H2O2 and mitophagy was assessed by Western blot of different mitochondrial complex subunits [i.e., NDUFB8 (complex I), SDHB (complex II), MTCO2 (complex IV)]. Basal and phospho ATM, as well as GSNOR were used to check the efficiency of siRNA-mediated knock down. Vinculin was used as loading control. "
NCBI gene IDs:
"GSNOR: 128
ATM: 472"
Figure legend: " E. In the same experimental settings, mitophagy was also assessed by RT-qPCR relative quantitation of D-loop (selected as measure of mtDNA) normalized to genomic actin (gActin). Results shown are the means ± SD of n = 8 experiments ***p&lt;0.001; n.s., not significant. "
NCBI gene IDs:
"gActin: 
genomic actin: "
Figure legend: " F. U2OS cells were depleted of endogenous ATM by repeated transfections with shRNA and induced, by doxycycline incubation, to express ATMWT, ATM2RA, or ATMCL mutant. Where indicated, cells were further transfected with a GSNOR-coding vector and then treated for 4 h with 100 μM H2O2. Mitophagy was assessed by RT-qPCR relative quantitation of D-loop normalized to genomic actin (gActin). Results shown are the means ± SD of n = 6 experiments. *p&lt;0.05; ***p&lt;0.001. "
NCBI gene IDs:
"gActin: 
genomic actin: 
GSNOR: 128
ATM: 472"
Figure legend: " A, B. ATMWT U2OS cells were treated with 10 μM CCCP for 8 h. After treatment, cells were incubated with 5 μM 2',7'-H2DCF-DA (A) or MitoSox (B) to evaluate the production of H2O2 or mitochondrial superoxide, respectively. Values are shown as units of DCF or MitoSox fluorescence relative to untreated cells (arbitrarily set as 1) and represent the means ± SD of n = 3 independent experiments. ***p&lt; 0.001. "
NCBI gene IDs:
"ATM: 472"
Figure legend: " U2OS cells were depleted of endogenous ATM by repeated transfections with shRNA and induced, by doxycycline incubation, to express ATMWT, ATM2RA, or ATMCL mutant. Where indicated, cells were further transfected with a GSNOR-coding vector and then treated for 2 h with 10 μM CCCP. Mitophagy was evaluated by: (D) RT-qPCR relative quantitation of D-loop normalized to genomic actin (gActin). Results shown are the means ± SD of n = 4 experiments. *p&lt;0.05; **p&lt;0.01; ***p&lt;0.001. "
NCBI gene IDs:
"gActin: 
genomic actin: 
GSNOR: 128
ATM: 472"
Figure legend: " U2OS cells were depleted of endogenous ATM by repeated transfections with shRNA and induced, by doxycycline incubation, to express ATMWT, ATM2RA, or ATMCL mutant. Where indicated, cells were further transfected with a GSNOR-coding vector and then treated for 2 h with 10 μM CCCP. (E fluorescence microscopy analyses upon incubation with chloroquine (CLQ) to enhance differences in mitophagy. Anti-TOM20 (red) was used to visualize mitochondria; anti-LC3 (green) was used to identify autophagosomes. "
NCBI gene IDs:
"GSNOR: 128
ATM: 472"
Figure legend: " U2OS cells were depleted of endogenous ATM by repeated transfections with shRNA and induced, by doxycycline incubation, to express ATMWT, ATM2RA, or ATMCL mutant. Where indicated, cells were further transfected with a GSNOR-coding vector and then treated for 2 h with 10 μM CCCP. F) fluorescence microscopy analyses upon incubation with chloroquine (CLQ) to enhance differences in mitophagy. Percentage of mitochondria merging with LC3-positive puncta calculated by Fiji analysis software using the open-source plugin ComDet v. 0.3.7. Values are expressed as % of mitochondria (TOM20+ particles) co-localizing with LC3/cell and graphed as boxes (25th-75th interquartile range) and whiskers (minimum to maximum showing all points), with central bands representing the median of n ≥ 9 different cells. **p&lt;0.01; ***p&lt;0.001; n.s., not significant. "
NCBI gene IDs:
"GSNOR: 128
ATM: 472"
Figure legend: " U2OS cells were depleted of endogenous ATM by repeated transfections with shRNA and induced, by doxycycline incubation, to express ATMWT, ATM2RA, or ATMCL mutant. Where indicated, cells were further transfected with a GSNOR-coding vector and then treated for 2 h with 10 μM CCCP. (G Western blot of different subunits of mitochondrial proteins, i.e., NDUFB8 (complex I), SDHA and SDHB (complex II), MTCO2 (complex IV) and voltage-dependent anion channel (VDAC). Tubulin, LDH and Vinculin were used as loading controls. "
NCBI gene IDs:
"GSNOR: 128
ATM: 472"
Figure legend: " U2OS cells were depleted of endogenous ATM by repeated transfections with shRNA and induced, by doxycycline incubation, to express ATMWT, ATM2RA, or ATMCL mutant. Where indicated, cells were further transfected with a GSNOR-coding vector and then treated for 2 h with 10 μM CCCP. H) Densitometry of mitochondrial protein immunoreactive bands and expressed as arbitrary units. Values shown represent the means ± SD of n = 3 independent experiments. *, p&lt;0.05; **, p&lt;0.01; ***, p&lt;0.001; n.s., not significant. "
NCBI gene IDs:
"GSNOR: 128
ATM: 472"
Figure legend: " B. HEK293 cells overexpressing the wild type form of GSNOR (GSNORwt) or an empty vector (Empty) were subjected to combined treatment (200 μM H2O2 + 400 μM DPTA) in the presence or absence of the ATM inhibitor KU55933 (KU). Analysis of dead cells were performed with Trypan blue exclusion assay. Western blot analysis of GSNOR is shown as inset in the graph to substantiate transfection efficiency. Data, shown as fold change of dead cells relative to untreated cells (arbitrarily set to 1), represent the mean count ± SEM of n = 4 experiments done in duplicate *p&lt;0.05; with respect to Empty cells. "
NCBI gene IDs:
"GSNOR: 128"
Figure legend: " C. Representative optic microscopy image of GSNORwt and Empty cells upon 24 h treatment with H2O2 + DPTA in the presence KU. "
NCBI gene IDs:
"GSNOR: 128"
Figure legend: "D. Western blot analysis of Caspase3 and PARP1 in GSNORwt and Empty cells treated as in panel C. Tubulin was used as loading control."
NCBI gene IDs:
"GSNOR: 128"
Figure legend: " E. HEK293 cells overexpressing the wild type form of GSNOR (GSNORwt) or an empty vector (Empty) were transfected with siRNA against Pakin (siParkin) or control siRNA (scramble, siScr). Afterwards, they were subjected to combined treatment (200 μM H2O2 + 400 μM DPTA) and viability assessed by Alamar blue (AB) fluorescent assay. Data, shown as fold change of viable cells, refer to AB fluorescence (relative to untreated cells, arbitrarily set to 1) and represent the means ± SD of n = 6 independent experiments. **, p&lt;0.01; n.s., not significant. "
NCBI gene IDs:
"GSNOR: 128
Pakin: 5071
Parkin: 5071"
Figure legend: " F. Western blot of Gsnor was assessed in mouse adult fibroblasts (MAFs), obtained from wild type (WT) and Gsnor-null (KO) mice treated with 200 or 400 μM H2O2 for 24 h. "
NCBI gene IDs:
"Gsnor: 11532"
Figure legend: " WT and Gsnor KO MAFs were subjected to treatment with 200 μM H2O2, or 400 μM DPTA, or a combination of both. Cell viability was evaluated by LIVE/DEAD assay. "
NCBI gene IDs:
"Gsnor: 11532"
Figure legend: " H. WT and Gsnor KO MAFs were subjected to treatment with 200 μM H2O2, or 400 μM DPTA, or a combination of both. Cell viability was evaluated by LIVE/DEAD assay. Scale bar = 50 µm. Data, shown as % of dead (red) cells, represent the mean count ± SD of n = 3 different fields of three independent experiments. *p&lt;0.05; n.s., not significant with respect to WT MAFs. "
NCBI gene IDs:
"Gsnor: 11532"
Figure legend: "A, B. HeLa cells treated with non-targeting siRNA (Ctrl) or C9orf72 siRNA and transfected with mCherry-EGFP-LC3 were treated with vehicle (Ctrl), Torin1 (250 nM; 3 h), bafilomycin A1 (100 nM; 6 h - BafA1) or combinations thereof as indicated. Autophagosomes (green+red) and autolysosomes (red only) were quantified per cell (Mean ± SEM from 3 independent experiments; one-way ANOVA with Fisher's LSD test: ns, not significant, * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001; N (cells) = Ctrl/Ctrl: 120; Ctrl/Torin1: 101; C9orf72/Ctrl: 99; C9orf72/Torin1: 106; Ctrl/BafA1: 116; Ctrl/Torin1/BafA1: 118; C9orf72/BafA1: 109; C9orf72/Torin1/BafA1: 106). Scale bar = 20 µm. C9orf72 knockdown was confirmed by RT-qPCR (Appendix Fig S2)."
NCBI gene IDs:
"C9orf72: 203228"
Figure legend: "C, D. HEK293 cells treated with non-targeting (Ctrl) or C9orf72 siRNA were incubated with BafA1, BafA1+Torin1 (C) or BafA1+rapamycin (D) and levels of LC3-I and II were determined on immunoblots. Levels of LC3-II were normalized against α-tubulin and are shown relative to the BafA1-treated sample (Mean ± SEM; one-way ANOVA with Fisher's LSD test: ns, not significant, * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001; N = 3 experiments)."
NCBI gene IDs:
"C9orf72: 203228"
Figure legend: "E, F. Primary cortical neurons (DIV5/6) were transfected with ECFP non-targeting (Ctrl) or C9orf72 miRNA (cyan) and EGFP-LC3 (green). 3 days post transfection neurons were treated with vehicle (Ctrl), Torin1 (250 nM; 3 h), BafA1 (100 nM; 5 h) or combinations thereof as indicated. Autophagosomes were quantified as the number of EGFP-LC3 positive puncta per soma from 2 independent experiments (Mean ± SEM; one-way ANOVA with Fisher's LSD test: ns, not significant, * p ≤ 0.05; *** P ≤ 0.001; **** P ≤ 0.0001; N (cells) = Ctrl miRNA/Ctrl: 77; Ctrl miRNA/Torin1: 68; C9orf72 miRNA/Ctrl: 57; C9orf72 miRNA/Torin1: 69; Ctrl miRNA/BafA1: 35; Ctrl miRNA/Torin1/BafA1: 57; C9orf72 miRNA/BafA1: 66; C9orf72 miRNA/Torin1/BafA1: 64). Scale bar = 5 µm."
NCBI gene IDs:
"C9orf72: "
Figure legend: "A. HEK293 cells treated with non-targeting (Ctrl) or FIP200 siRNA were co-transfected with EGFP-LC3 and either empty vector control (EV), Myc-C9orf72S or Myc-C9orf72L. 24 h post transfection cells were treated with vehicle or 100 nM BafA1 for 6 h. Samples were lysed and subjected to SDS-PAGE and immunoblot. Autophagy levels were determined by immunoblot for EGFP-LC3-I and II. Expression of Myc-C9orf72 was confirmed using anti-Myc (* indicates a nonspecific band). FIP200 knockdown was confirmed using anti-FIP200 antibodies. α-tubulin was used as loading control."
NCBI gene IDs:
"C9orf72: 203228
FIP200: 9821"
Figure legend: "B. HeLa cells treated with non-targeting (Ctrl) or FIP200 siRNA were co-transfected with empty vector (EV), Myc-C9orf72S or Myc-C9orf72L (red) and EGFP-LC3 (green) to label autophagosomes. As positive control EV transfected cells were treated for 3 h with Torin1 (250 nM). Autophagy was quantified as the number of EGFP-LC3 positive autophagosomes per cell from 3 independent experiments (Mean ± SEM; one-way ANOVA with Fisher's LSD test: **** P ≤ 0.0001, N (cells) = Ctrl/EV: 73; FIP200/EV: 76; Ctrl/EV/Torin1: 73, FIP200/EV/Torin1: 70; Ctrl/C9orf72L: 71; FIP200/C9orf72L: 74; Ctrl/C9orf72S: 72; FIP200/C9orf72S: 72). Scale bar = 20 µm. FIP200 knockdown was confirmed by immunoblot (Appendix Fig S2)."
NCBI gene IDs:
"C9orf72: 203228
FIP200: 9821"
Figure legend: "A, B, C. Cell lysates of HEK293 cells co-transfected with FLAG-FIP200 (A) and either empty vector control, Myc-C9orf72S or Myc-C9orf72L were subjected to immunoprecipitation with anti-Myc (A and B) antibodies. Immune pellets were probed for Myc-C9orf72 (A and B), FLAG-FIP200 (A) on immunoblots."
NCBI gene IDs:
"C9orf72: 203228"
Figure legend: "A, B, C. Cell lysates of HEK293 cells co-transfected with HA-ULK1 (B) and either empty vector control, Myc-C9orf72S or Myc-C9orf72L were subjected to immunoprecipitation with anti-Myc (A and B) antibodies. Immune pellets were probed for Myc-C9orf72 (A and B), HA-ULK1 (B) on immunoblots."
NCBI gene IDs:
"C9orf72: 203228"
Figure legend: "A, B, C. Cell lysates of HEK293 cells co-transfected with Myc-ATG13 (C) and either empty vector control, FLAG-C9orf72S or FLAG-C9orf72L were subjected to immunoprecipitation with anti-FLAG (C) antibodies. Immune pellets were probed for FLAG-C9orf72 (C), Myc-ATG13 (C) on immunoblots."
NCBI gene IDs:
"C9orf72: 203228"
Figure legend: "D, E. Cell lysates of HEK293 cells transfected with empty vector control, Myc-C9orf72S or Myc-C9orf72L were subjected to immunoprecipitation with anti-Myc antibodies Immune pellets were probed for Myc-C9orf72 and endogenous FIP200, ULK1, and ATG13. There are multiple alternatively spliced forms of ATG13 (Jung et al., 2009; Alers et al., 2011); ATG13* is most likely a smaller alternative spliced form of ATG13 that is enriched by interaction with C9orf72."
NCBI gene IDs:
"C9orf72: 203228"
Figure legend: "F, G, H. HeLa cells transfected with Flag-FIP200 and Myc-C9orf72 were fixed and processed for PLA analysis. Transfections were laced with mVenus to enable identification of transfected cells for analysis where required (green). PLA signals (red) were counted per cell (Mean ± SEM; one-way ANOVA with Fisher's LSD test, **** P ≤ 0.0001; N (cells) = (F) C9orf72S: 22; C9orf72L: 18; EV+FIP200: 18; C9orf72S+FIP200: 18; C9orf72L+FIP200: 17. Scale bar = 10 µm; see also Fig EV2."
NCBI gene IDs:
"FIP200: 9821"
Figure legend: "F, G, H. HeLa cells transfected with HA-ULK1 and Myc-C9orf72 were fixed and processed for PLA analysis. Transfections were laced with mVenus to enable identification of transfected cells for analysis where required (green). PLA signals (red) were counted per cell (Mean ± SEM; one-way ANOVA with Fisher's LSD test, **** P ≤ 0.0001; N (cells) = (G) C9orf72S: 21; C9orf72L: 20; EV+ULK1: 20; C9orf72S+ULK1: 22; C9orf72L+ULK1: 20. Scale bar = 10 µm; see also Fig EV2."
NCBI gene IDs:
"ULK1: 8408"
Figure legend: "F, G, H. HeLa cells transfected with Myc-ATG13 and EGFP-C9orf72 were fixed and processed for PLA analysis. Transfections were laced with mVenus to enable identification of transfected cells for analysis where required (green). PLA signals (red) were counted per cell (Mean ± SEM; one-way ANOVA with Fisher's LSD test, **** P ≤ 0.0001; N (cells) = (H) C9orf72S: 11; C9orf72L: 10; EV+ ATG13: 11; C9orf72S+ATG13: 11; C9orf72L+ATG13: 11). Scale bar = 10 µm; see also Fig EV2."
NCBI gene IDs:
"ATG13: 9776"
Figure legend: "A. HEK293 cells were transfected with non-targeting (Ctrl) or C9orf72 siRNA. Cells were treated with rapamycin for 6 h to induce autophagy. Activation of ULK1 was determined on immunoblots using phospho-ULK1 (Ser757), total ULK1 and GAPDH Abs (loading control)."
NCBI gene IDs:
"C9orf72: 203228"
Figure legend: "B. HeLa cells treated with non-targeting (Ctrl) or C9orf72 siRNA were transfected with mCherry-FIP200. 24 h post transfection cells were treated for 3 h with Torin1 (250 nM) or vehicle (Ctrl). Translocation of the ULK1 complex was quantified as the number of mCherry-FIP200 positive puncta per cell from 3 independent experiments (Mean ± SEM; one-way ANOVA with Fisher's LSD test, ns: not significant, **** P ≤ 0.0001; N (cells) = Ctrl/Ctrl: 65; Ctrl/Torin1: 60; C9orf72/Ctrl: 54; C9orf72/Torin1: 49). C9orf72 knockdown was determined by RT-qPCR (Appendix Fig S2). Scale bar = 10 µm."
NCBI gene IDs:
"C9orf72: 203228"
Figure legend: "C. Primary cortical neurons (DIV5/6) were transfected with EmGFP non-targeting (Ctrl) or C9orf72 miRNA (green) and mCherry-FIP200 (red); for rescue experiments the cells were additionally transfected with mCerulean-tagged C9orf72s and C9orf72L (cyan). 3 days post transfection neurons were treated for 3 h with Torin1 (250 nM) or vehicle (Ctrl). Translocation of the ULK1 complex was quantified as the number of mCherry-FIP200 positive puncta per soma from 2 independent experiments (Mean ± SEM; one-way ANOVA with Fisher's LSD test, ns: not significant, *** P ≤ 0.001, **** P ≤ 0.0001; N (cells) = Ctrl miRNA/Ctrl: 134; Ctrl miRNA/Torin1: 125; C9orf72 miRNA/Ctrl: 101; C9orf72 miRNA/Torin1: 78; C9orf72 miRNA+C9orf72L+C9orf72S: 41; C9orf72 miRNA+C9orf72L+C9orf72S/Torin1: 39). Scale bar = 5 µm."
NCBI gene IDs:
"C9orf72: "
Figure legend: "D. HeLa cells were co-transfected with mCherry-FIP200 (red) and empty vector (EV), FLAG-C9orf72L or FLAG-C9orf72S (green). As positive control EV transfected cells were treated for 3 h with Torin1 (250 nM). Translocation of the ULK1 complex was quantified as the number of mCherry-FIP200 positive puncta per cell from 3 independent experiments (Mean ± SEM; one-way ANOVA with Fisher's LSD test, *** P ≤ 0.001, **** P ≤ 0.0001); N (cells) = EV: 47, EV + Torin1: 31, C9orf72L: 46, C9orf72S: 45). Scale bar = 10 µm."
NCBI gene IDs:
"C9orf72: 203228"