Extended Data Fig. 5: Valine deprivation promotes the DNA damage response.
From: Human HDAC6 senses valine abundancy to regulate DNA damage
a, Immunoblot of DNA damage marker γH2AX and PARylation (PAR) in HCT116 cells upon valine deprivation with different time points. b, c, Representative images (b) and quantification (c) with immunofluorescence staining for γH2AX (red) in HCT116 cells upon valine deprivation for 24 h. Scale bar, 10 μm. d, Immunoblot of DNA damage marker γH2AX and PARylation (PAR) in HCT116 cells cultured with different concentrations of valine for 48 h. e, f, Representative images (e) and quantification (f) with immunofluorescence staining for γH2AX (red) in HCT116 cells upon valine deprivation for 48 h at different concentrations. Scale bar, 10 μm. g, Representative comet assay (top) and quantification (bottom) showing the tail moment of HCT116 cells upon valine deprivation for 24 h. h, Representative comet assay (top) and quantification (bottom) showing the tail moment of HCT116 cell line upon valine deprivation for 48 h at different concentrations. i, Immunoblot of DNA damage marker γH2AX and PARylation (PAR) in HDAC6 and TET2 knockdown HCT116 cell line upon valine deprivation at different concentration with 48 h. j, Schematic of programmed breaks in active DNA demethylation via TET2-TDG axis. k, The levels of 5fC upon valine deprivation in WT, TDG knockdown HCT116 cell lines via UPLC–MS/MS. l, The 5caC and 5fC levels in genome upon valine deprivation in WT, TDG knockdown HCT116 cell lines via Dot-blot assay. m, Average plots (top) and heatmaps (bottom) showing the enrichment of END-seq and ddC-S1-END-seq signals in HCT116 cell line upon valine deprivation at the valine deprivation-specific enhanced TET2 binding regions. “0”, peak centre. n, Flow cytometry analysis of cell cycle upon valine deprivation for 24 h with the pretreatment of Palboclib (PLB, PD0332991) for 24 h in HCT116 cell lines. o, The 5hmC, 5caC and 5fC levels in genome upon valine deprivation for 24 h with the pretreatment of Palboclib (PLB, PD0332991) for 24 h in HCT116 cell lines via dot-blot assay. p, q, Representative images (p) and quantification (q) with immunofluorescence staining for γH2AX (red) and 53BP1 (green) in WT or Tdg knockdown HCT116 cell line upon pretreatment with Palboclib (PLB, PD.332991) for 24 h and then valine deprivation for 24 h. Scale bar, 10 µm. r, The overlap of differentially expressed genes (DEGs) in WT, HDAC6 knockdown and TET2 knockdown HCT116 cells upon valine deprivation. s, Among the 177 valine-modulated and HDAC6-TET2 axis dependent genes, the change expression of DNA damage and repair-related genes in three types of HCT116 cell lines upon valine deprivation. t, GO analysis of the 177 valine-modulated and HDAC6-TET2 axis dependent genes in r. u, v, RT–qPCR validation of a subset of DNA-damage and repair genes upon valine deprivation with 24 h (u) or at different valine concentration with 24 h (v). w, x, Flow cytometry analysis of cell cycle (w) and quantification (x). Schematic in j was created using BioRender (https://BioRender.com). For c, f, and q, n = 35 microscopic views examined across 3 independent experiments. For g, h, k, u, v and x, n = 3 independent experiments. Data are presented as mean ± s.d.. Statistical analysis was performed using Mann–Whitney U-test (c, f, g, h, q) and one-way ANOVA (k, u, v, x); *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, NS, not significant. For GO analysis, one-sided Fisher’s exact test P-values are applied to evaluate gene enrichment in annotation terms. For gel source data, see Supplementary Fig. 1.
