EMBO/SourceData huggingface dataset:
Figure 5C. IF for Cas9 and CENP-C of metaphases of RPE1-dCas9-Kin14VIb cells transduced with Chr1-telo sgRNA in the presence or absence of 500 nM rapalog. The Chr1-telo loci (Cas9 foci, white arrowheads) are aligned on the metaphase plate in control condition (- rapalog), while the loci are facing either a single or opposite poles after rapalog addition to induce Kin14VIb binding to dCas9. Scale bar = 5 µm. For clarity, the maximum intensity projection of a subset of z stacks (18-50/100) is shown. Data information: (C for optimal visibility of the Chr1-telo foci, the brightness and contrast of all channels were linearly adjusted for individual cells.D, Live cell microscopy of asynchronously growing RPE1-dCas9-Kin14VIb cells transduced with Chr1-telo sgRNA in the presence or absence of 500 nM rapalog to induce kinesin binding to the subtelomeric locus. (D) Stills showing the most frequently observed metaphase localization and daughter cell distribution (white arrowheads) of the duplicated Chr1-telo loci. SiR-DNA was used to visualize the DNA, GFP depicts the Chr1-telo loci. Time (h:min). Scale bar = 5 µm. Note that in the upper panel, the cell was already in mitosis when imaging started (t =0:00). Data information: (D, Cells were derived from three independent imaging experiments. N = 23 cells (- rapalog), n = 41 cells (+ rapalog). Sp = same pole, op = opposite pole, ss = same sisters. D): for optimal visibility of the Chr1-telo foci, the brightness and contrast of all channels were linearly adjusted for individual cells.
Figure 6B, Live cell microscopy of asynchronously growing RPE1-dCas9-Kin14VIb cells transduced with Chr9-cen sgRNA and plus/minus rapalog to induce kinesin binding to the pericentromeric locus (Movies S4-S8). (B) Stills of the most frequently observed metaphase orientations and daughter cell distributions of the duplicated Chr9-cen loci (white arrowheads) are shown. SiR-DNA was used to visualize the DNA, GFP depicts the Chr9-cen loci. Time (h:min). Scale bar = 5 µm. Data information: Cells were derived from two independent imaging experiments.. In (B), for optimal visibility of the Chr9-cen foci, the brightness and contrast of all channels were linearly adjusted for individual cells. See Movies EV4- S8 for equally adjusted corresponding examples.D. Time between nuclear envelop break down (NEB) and anaphase onset in the presence (Kin14VIb bound to dCas9) or absence (Kin14VIb expressed, but not bound to dCas9) of rapalog. Data information: Cells were derived from two independent imaging experiments.. (D): Mean and S.D. are shown.
Figure 7A. Representative IF images of RPE1-dCas9-Kin14VIb cells, showing the localization of the Chr9-cen loci in metaphase in the absence or presence or rapalog. Magnifications of the white boxed regions (each region showing one Chr9-cen locus) are shown in the corners (scale bars = 2 µm). The fraction of cells with Chr9 mis-aligned and CENP-C near the locus (Cas9) is indicated below the image. For clarity, the maximum intensity projection of a subset of z stacks in which the Chr9-cen loci are in focus (2-10/100) is shown. B. Frequency of the observed metaphase localizations of the Chr9-cen loci as shown in (A). Data information: In all cases, bars represent the mean of 2 independent experiments, while dark- and light-colored dots represent the values of each experiment. (B): **** P<0.0001, Fisher's exact test. N ≥ 31 cells per condition, per experiment. (A, For optimal visibility of the Chr9-cen foci the brightness and contrast of all channels were linearly adjusted for individual cells.C. Representative IF images of RPE1-dCas9-Kin14VIb cells, showing the segregation and distribution of Chr9-cen loci in anaphase (white arrowheads). Red arrowheads indicate H3S10ph-positive chromatin bridges. For clarity, the maximum intensity projection of a subset of z stacks in which the chr9-cen loci are in focus (30-62/100) is shown. Scale bar = 2 µm. Data information: C): For optimal visibility of the Chr9-cen foci H3S10ph-positive chromatin bridges, the brightness and contrast of all channels were linearly adjusted for individual cells.E. Frequency of cells with a 2-2 distribution of the Chr9-telo loci. Data information: In all cases, bars represent the mean of 2 independent experiments, while dark- and light-colored dots represent the values of each experiment. : **** P<0.0001, Fisher's exact test. N ≥ 29 cells per condition, per experiment.F. Frequency of the observed segregation/distribution errors of the Chr9-cen loci as shown in (C). Data information: In all cases, bars represent the mean of 2 independent experiments, while dark- and light-colored dots represent the values of each experiment. F): **** P<0.0001, Fisher's exact test. N ≥ 29 cells per condition, per experiment.
Figure 8C. Average percentage of the bases of the p or q arm that are (classified as) 0, 1, 3 or 4-somy. Percentages are depicted as stacked bar plots and are determined for all copy numbers other than 2-somy (euploid).
Figure 1E Distribution of endogenous WIPI2 and STING in MEFs. F Statistical analysis of the number of STING- and WIPI2-positive puncta in cells treated as in (E). G Distribution of endogenous ATG16L1 in HEK293T cells stably expressing STING-HA. H Statistical analysis of the number of STING- and ATG16L1-positive puncta in cells treated as in (G). All the cells were treated with or without cGAMP for 2 h. All statistical data are presented as mean ± SEM of three independent experiments.
Figure 2A Co-immunoprecipitation of endogenous STING with WIPI1/2-Myc in MEFs transfected with Myc-tagged WIPI1 or WIPI2. The cells were treated with or without cGAMP for 2 h and the WIPI1/2-Myc precipitates were analyzed by western blot using anti-STING. B Co-immunoprecipitation of endogenous WIPI2 with HA-tagged STING, STING-R238A or STING-S366A from HEK293T cells stably expressing STING-HA STING-R238A-HA or STING-S366A-HA. The cells were treated with or without cGAMP for 2 h.Co-immunoprecipitation of endogenous STING with Myc- WIPI2 or the indicated WIPI2 mutants from MEFs transfected with the indicated plasmids.J Purified GST-tagged STING-153-379 or STING-153-379-3A-2 was incubated with recombinant WIPI2 or the indicated WIPI2 mutants, and then precipitated with glutathione sepharose beads. The bound WT or mutant WIPI2 was detected by western blot using anti-WIPI2. K Statistical analysis of (J).
Figure 3A WIPI2-GFP puncta in MEFs stably expressing GFP-tagged WT or mutant WIPI2. The cells were treated with cGAMP for 2 h or Torin1 for 1 h. B Statistical analysis of the number of WIPI2-GFP puncta in cells treated as in (A).F LC3 and p62 level in HEK293T cells stably expressing HA-tagged WT or mutant STING. The cells were treated with or without cGAMP for 4 h.
Figure 4C Co-immunoprecipitation of endogenous WIPI2 with STING-HA from HEK293 cells stably expressing STING-HA with or without BECN1 deletion. The cells were treated with or without Torin1 for 3 h.E Co-immunoprecipitation of endogenous STING with Myc-tagged WT or mutant WIPI2 from MEFs treated with or without VPS34-IN1 for 2 h.G GFP-LC3 punctum formation in MEFs stably expressing GFP-LC3 and STING-HA. The cells were treated with or without cGAMP or VPS34-IN1 for 2 h. Scale bars, 10 µm.I Western blot assay of p62 and phospho-TBK1 (Ser172) in MEFs. The cells were pretreated with or without VPS34-IN1 for 1 h and then co-treated with cGAMP or Torin1 for another 4 h along with or without CQ. CQ, chloroquine. J Statistical analysis of p62 protein level in cells treated as in (I).
Figure 5A, B Localization of endogenous WIPI2, WIPI1, ATG16L1 (A) or Myc-tagged WT WIPI2 or WIPI2 mutants (B) in MEFs transfected with Cy3 labeled-ISD (1 μg/ml). ISD, interferon stimulatory DNA.D Statistical analysis of cytoplasmic DNA puncta in cells treated as in (C).F Statistical analysis of cytoplasmic DNA puncta in cells treated as in (E).
Figure 6A Wipi2- or Sting-deleted MEFs were treated with Ara-C for 12 h, and then cultured in Ara-C-free medium. After 24 h, the cells were lysed and analyzed by western blot using the indicated antibodies. B Statistical analysis of the protein level of phospho-TBK1 (Ser172) and STING in cells treated as in (A). The relative phospho-TBK1 level was normalized to TBK1, the relative STING level was normalized to Actin.C-E MEFs with Wipi2 or Sting deletion (C), Wipi2-KO MEFs with transfection of Myc-tagged WT or mutant WIPI2 (D), or Sting-KO MEFs with transfection of HA-tagged WT or mutant STING (E), were treated with Ara-C for 12 h and then cultured in Ara-C-free medium for another 24 h. Total mRNA was extracted and the Ifnb1 mRNA level was measured by real-time PCR and normalized to Actb mRNA. 2RE, WIPI2-R108E/R125E.F Sting-KO MEFs with transfection of HA-tagged WT or mutant STING were treated with cGAMP for the indicated time. The cells were lysed and analyzed by western blot using the indicated antibodies.
Figure 1Dose-response curve of HF and DWN12088 in WI-26 VA4 cells. The IC50 value for collagen level was measured as a representative of chemical efficacy. The cells were starved of serum for 6 hr and then incubated with the indicated concentrations of compounds in the presence of TGF-β (2 ng/ml) for 72 hr. The secreted pro-collagen I levels were determined by ELISA. The CC50 values were determined by CellTiter-Glo assay. Dose-response curves were determined by combining at least 2 biologically independent experiments. The IC50 values were calculated using GraphPad Prism 7.0 (Collagen level, n = 5 from three independent experiments (singlicate for one experiment and duplicate for two experiments; mean ± SEM); Celltiter-Glo assay, n = 4 from two independent experiments (duplicate for each experiment; mean ± SEM). ; HF (0.05), HF 0.05 mg/kg; HF (0.1), HF 0.1 mg/kg; DWN12088 (10), DWN12088 10 mg/kg;In vivo efficacy of HF and DWN12088 was determined in a bleomycin-induced lung fibrosis model. The indicated concentrations of HF or DWN12088 were orally administered to mice once a day for two weeks from a week after intratracheal injection of bleomycin (D). The saturation of percutaneous oxygen (SpO2) (E) was determined as a measurement of lung function. BLM, bleomycin; ; HF (0.05), HF 0.05 mg/kg; HF (0.1), HF 0.1 mg/kg; DWN12088 (10), DWN12088 10 mg/kgIn vivo efficacy of HF and DWN12088 was determined in a bleomycin-induced lung fibrosis model. The indicated concentrations of HF or DWN12088 were orally administered to mice once a day for two weeks from a week after intratracheal injection of bleomycin The collagen level was determined by hydroxyproline assay (G) BLM, bleomycin HF (0.05), HF 0.05 mg/kg; HF (0.1), HF 0.1 mg/kg; DWN12088 (10), DWN12088 10 mg/kg;In vivo efficacy of HF and DWN12088 was determined in a bleomycin-induced lung fibrosis model. The indicated concentrations of HF or DWN12088 were orally administered to mice once a day for two weeks from a week after intratracheal injection of bleomycin (D). Masson's trichrome staining of lung tissues. ; HF (0.05), HF 0.05 mg/kg; HF (0.1), HF 0.1 mg/kg; DWN12088 (10), DWN12088 10 mg/kg; b, bronchiole; v, blood vessel.
Figure 2WI-26 VA4 cells were incubated in the presence of the indicated concentrations of HF and DWN12088 for 72 hr. The chemical concentrations were determined based on the IC50 for collagen level (HF, 15 nM; DWN12088, 2 μM). The levels of proteins were determined by immunoblot assay. The proline content of the proteins based on Uniprot amino acid sequence is shown on the right side of the immunoblot images.WI-26 VA4 cells were incubated with the indicated concentrations of HF and DWN12088 in the presence of 2 ng/ml TGF-β for 15 hr. The chemical concentrations were determined based on the IC50 values for collagen levels (HF, 15 nM; DWN12088, 2 μM). The activation of the TGF-β pathway was determined by monitoring the phosphorylation of SMAD2. Immunoblot images are representative of three biologically independent experiments.   The band intensities of the immunoblot images in (C) were quantified using ImageJ (n = 3; One-way ANOVA; *P < 0.05, **P < 0.01, ***P < 0.001; mean ± SEM).
Figure 5The IC50 values of the indicated compounds were determined for collagen level (HF and DWN12088, n = 8 from four independent experiments (duplicate for each experiment); DWN11748, n = 12 from six independent experiments (duplicate for each experiment); DWN11761 and DWN11251, n = 6 from three independent experiments (duplicate for each experiment); mean ± SEM). The IC50 values of the indicated compounds were determined for cytotoxicity (n = 4 from two independent experiments (duplicate for each experiment); mean ± SEM). The IC50 values of the indicated compounds were determined for TNT-luciferase activity (n = 6 from two independent experiments (triplicate for each experiment); only for DWN11761, n = 9 from three independent experiments (triplicate for each experiment); mean ± SEM). The IC50 values of the indicated compounds were determined for puromycin incorporation (HF, n = 12 from four independent experiments (triplicate for each experiment); DWN11748, DWN12088, and DWN11251, n = 9 from three independent experiments (triplicate for each experiment); DWN11761, n = 8 from three independent experiments (triplicate for two experiments and duplicate for one experiment); mean ± SEM).
Figure 6The binding of HF and DWN12088 to in vitro purified PARS1 WT and F1097A/E1123A/R1152L (FA/EA/RL) mutant was determined via microscale thermophoresis (MST). Kd values are listed in the top left part of the graph (technical replicates n = 3; mean ± SD).Catalytic activities of PARS1 WT and FA/EA/RL mutant were determined by prolylation assay in the presence of [3H]proline. To monitor the effect of PARS1 RA/EA/RL mutant on the activity of PARS1 WT, the same concentrations of the proteins were pre-incubated for 10 min and then used for the assay. The formation of Pro-tRNAPro was measured using liquid scintillation counter (technical replicates n = 3; mean ± SEM).293T cells were transfected with the indicated plasmid DNAs for 24 hr. The cell lysates were prepared and pulled down with Strep bead for 2 hr. Co-precipitated proteins were determined by immunoblot assay. WT, PARS1 wild type; MT, PARS1 FA/EA/RL.G, H. WI-26 VA4 cells stably expressing each of EV (empty vector) or PARS1 FA/EA/RL mutant were incubated with the indicated compounds for 72 hr. Dose-response curves of the compounds for collagen levels and cytotoxicity were determined and relative ratio of CC50 to the IC50 of collagen for HF (G) and DWN12088 (H) were calculated. The IC50 values were calculated using GraphPad Prism 7.0 (collagen ELISA, n = 12 from four independent experiments (triplicate for each experiment); cytotoxicity assay, n = 8 from four independent experiments (duplicate for each experiment); Welch's t test; ***P < 0.001; mean ± SEM).K. WI-26 VA4 cells stably expressing each of EV or PARS1 FA/EA/RL mutant were incubated with the indicated compounds in the presence of TGF-β for 72 hr. The levels of the indicated proteins were determined by immunoblotting. The percentage of proline in polypeptide sequences is shown on the right side of immunoblot images.
Figure 1(E and F) HepG2 cells infected with lentivirus expressing p53 sgRNA or control sgRNA were treated with ethanol-d6 (5 mM) or ethanol-1-13C (5 mM) for 0.5 h. Relative acetate m+3 and acetate m+1 levels (E-F, top panels) were examined. Protein levels of p53 and ALDH2 (E-F, bottom panels) were analyzed by western blot.(H-J) HepG2 cells infected with lentivirus expressing p53 sgRNA or control sgRNA were treated with doxorubicin (2 ng/mL) (H) nutlin3α (10 μM) (I) or cyanamide (1 mM) (J) for 24 h. Relative enzymatic activity of ALDH2 was examined (top panel). Protein expression was determined by western blot analysis (bottom panel).(P-R) p53+/+ and p53-/- mice were injected intraperitoneally with ethanol-d6 (2 g/kg) for 0.5 h. Relative acetate m+3 levels in mouse liver tissues (P) and mouse serum (Q), and relative hepatic acetyl-CoA (R) were examined.(S) Mouse hepatocyte isolated from p53+/+ and p53-/- mice and lysates were treated with or without cyanamide (1mM). Relative acetyl-CoA level (top panel) and protein expression (bottom panel) were measured. (T) CCC-HEL-1 cells transfected with control siRNA or p53 siRNA were treated with or without cyanamide (1mM). Relative acetyl-CoA level (top panel) and protein expression (bottom panel) were analyzed.(U-X) HepG2 cells infected with lentivirus expressing p53 sgRNA or control sgRNA were transfected with control siRNA or ALDH2 siRNA and were treated with ethanol-d6 (5 mM) (U) or ethanol-1-13C (5 mM) (X) for 0.5 h. (U) Relative acetate m+3 and acetyl-CoA m+3 levels are shown. (X) Relative acetate m+1 and acetyl-CoA m+3 levels were examined. Protein levels of p53 and ALDH2 were analyzed by western blot (V- W).
Figure 2(C) Lysates from mice liver tissues (WT, p53-/- and ALDH2-/-) were immunoprecipitated using p53 or ALDH2 antibody, and bound proteins were analyzed by western blot.(G) HepG2 cells were infected with lentivirus expressing ALDH2 sgRNA or control sgRNA and overexpressed hygromycin-resistant ALDH2 mut312-323 in ALDH2-knockout cell lines. The cells were then transfected with control siRNA or p53 siRNA for 48 h and treated with ethanol (5 mM) for 0.5 h and relative ALDH2 enzymatic activities were examined. Protein expression was analyzed by western blot.(H) Lysates from 293T cells transfected with HA-ALDH2, Flag-ALDH2 and increasing amounts of GST-p53 or vector control (-) as indicated were immunoprecipitated using anti-Flag M2 affinity gels, and bound proteins were analyzed by western blot.(I) HepG2 cells infected with lentivirus expressing ALDH2 sgRNA or control sgRNA were treated with ethanol (5 mM) for 0.5 h and cell lysates were treated with disuccinimidyl suberate (DSS) or DMSO for 0.5 h and protein levels of p53 and ALDH2 were examined.(J) Subcellular fractionation of HepG2 cells treated with doxorubicin (2 ng/mL) or nutlin3α (10 μM) or PFTα (10 μM) for 24 h were immunoprecipitated using p53 antibody, and bound proteins were analyzed by western blot.
Figure 3(A-B) HepG2 cells infected with lentivirus expressing p53 sgRNA or control sgRNA under glucose or pyruvate deprivation and cell lysates were used to measure ALDH2 activities (A, B, top panels) and examine p53 and ALDH2 proteins (A, B, bottom panels).(E) CETSAs exhibit the binding affinity of pyruvate to the N186 amino acid in HepG2 cells. Cell lysates were separated by SDS-PAGE and analyzed by western blot. Actin was used as an internal control. Relative ALDH2 band intensities were quantified and plotted against corresponding incubation temperatures.(F) HepG2 cells were infected with lentivirus expressing ALDH2 sgRNA or control sgRNA and overexpressed hygromycin-resistant ALDH2-N186A, E285A, C319A in ALDH2-knockout cell lines. Cells were digested and cell lysates were incubated with the indicated doses of pyruvate for 1 hour on ice and 0.5 hour at room temperature, followed by pronase digestion. ALDH2 proteins were analyzed by western blot. Relative ALDH2/Actin ratios are shown.(H-J) HepG2 cells were infected with lentivirus expressing ALDH2 sgRNA or control sgRNA and overexpressed hygromycin-resistant ALDH2 N186A in ALDH2-knockout cell lines. The cells were treated with pyruvate (1 mM) for 24 h. Relative ALDH2 activity (H, top panel), acetate (I) and acetyl-CoA (J) were measured. The expression of p53 and ALDH2 were analyzed by western blot (H, bottom panel).
Figure 4H&E (hematoxylin-eosin) staining (C, top) and oil red O staining (C, bottom) were examined.Relative mRNA levels of IL-6, were analyzed. were examined. Data in are from n=5 mice per group.Relative mRNA levels of , IL-1β, TNF-α, Cxcl1, Cxcl2, Col1a1 were analyzed. Data in are from n=5 mice per group.(J) Relative triglyceride level was examined. (K-L) Relative levels of serum ALT (K) and AST (L) were examined. Data in are from n=5 mice per group.(M-O) WT, p53−/−, ALDH2−/− and p53−/−ALDH2−/− C57BL/6N male mice were injected intraperitoneally with ethanol-d6 (2 g/kg) for 0.5 h as shown in Figure 1N. Relative acetate and acetate m+3 levels in mouse liver tissues (M) and mouse serum (N) were examined. (O) Relative acetyl-CoA and acetyl-CoA m+3 levels in mouse liver tissues were measured.
Figure 5(C-D) p53+/+ and p53-/- mice were injected intraperitoneally with ethanol-d6 (2 g/kg) for 1 h. Chromatin fractions of liver tissues were subjected to ChIP-seq assay using H3K9ac antibody (left panel), H3K27ac antibody (right panel). Heat map of H3K9ac/H3K27ac binding signal (rows) from -1 kb to +1 kb surrounding the centre of H3K9ac/H3K27ac-binding sites for ChIP-seq tags (C). Heatmap of H3K9ac/H3K27ac binding signal (rows) from -1 kb to +1 kb surrounding the centre of H3K9ac/H3K27ac-binding sites for ChIP-seq tags of fatty acid metabolism related genes (D). For ChIP-seq analysis , there were no technical replicates for anti-H3K9ac or anti-H3K27ac.(F) ChIP assays with H3K9ac antibody and H3K27ac antibody or IgG in WT and p53−/− mice injected intraperitoneally with ethanol-d6 (2 g/kg) or saline for 1 h. Bound SCD1 response elements (RE) were analyzed by qRT-PCR. Data in F are from n=3 biological independent samples.WT, p53−/−, ALDH2−/− and p53−/−ALDH2−/− C57BL/6N male mice were fed control diet and ethanol diet as shown in Fig.4A. Relative mRNA level of SCD1 (G, top panel) was analyzed. Protein levels of p53, ALDH2, SCD1, Histone H3 and Ac-H3, caspase 3, cleaved caspase 3, p21 (G, bottom panel) in mice liver tissues were examined. Data in G are from n=5 technical replicates from one of three independent experiments with similar results.
Figure 6WT and p53−/− mice were injected with adenovirus expressing SCD1 shRNA or control shRNA via tail vein. After 7 days, WT and p53−/− mice were treated as described in the methods to establish a mouse model of chronic-binge ethanol consumption. (C) Protein levels of p53 and SCD1 were examined by western blot. level were examined.WT and p53−/− mice were injected with adenovirus expressing SCD1 shRNA or control shRNA via tail vein. After 7 days, WT and p53−/− mice were treated as described in the methods to establish a mouse model of chronic-binge ethanol consumption. Relative hepatic triglyceride level(D), relative serum ALT (E, left panel) and AST (E, right panel) level were examined.WT, ALDH2−/− and p53-/-ALDH2−/− mice were injected with adeno-associated virus expressing AAV-GFP or AAV-SCD1 via tail vein. After 7 days, WT, ALDH2−/− and p53-/-ALDH2−/− mice were treated as described in the methods to establish a mouse model of chronic-binge ethanol consumption. (H) Protein levels of p53 and SCD1 are shown.WT, ALDH2−/− and p53-/-ALDH2−/− mice were injected with adeno-associated virus expressing AAV-GFP or AAV-SCD1 via tail vein. After 7 days, WT, ALDH2−/− and p53-/-ALDH2−/− mice were treated as described in the methods to establish a mouse model of chronic-binge ethanol consumption. Relative hepatic triglyceride level (I), relative serum ALT (J, left panel) and AST (J, right panel) were examined.
Figure 2A. Left: Snapshots of actin comet tails assembled in Assembly conditions: 4.5 µm polystyrene beads coated with 400 nM Strep-SNAP-WA; 3 µM actin, 6 µM profilin, 90 nM Arp2/3 complex, 15 nM capping protein. Right: Tracking of the comet shown in the snapshots. Time is encoded in color. B. Left: Snapshots of actin comet tails assembled in Disassembly conditions: same as assembly conditions with 200 nM ADF/cofilin added. Right: Tracking of the comet shown in the snapshots. Time is encoded in color. C. Left: Snapshots of actin comet tails assembled in Recycling conditions: Disassembly conditions with 400 nM Cyclase Associated Protein (CAP) added. Right: Tracking of the comet shown in the snapshots. Time is encoded in color.E. Estimation of half-life of bead motility (from an exponential fit to the bead velocity curve) for each independent dataset in the different dynamic conditions (1 point per independent dataset). Data information: Mean and standard deviation are represented. Assembly: n = 20 comet tails. Disassembly: n = 13 comet tails. Recycling: n = 16 comet tails.