Fig. 5

Depletion of Trim66 results in the retention of the level of H3K56ac following DNA damage. a Immunoblot analysis of H3K56ac in shTrim66 tet-on ESCs as described in Fig. 3c. b Representative immunofluorescence images (left) and quantification (right) of H3K56ac in WT and TRIM66-KO ESCs at different time points after treatment with etoposide (EPI, 10 μM for 30 min). Scale bar, 10 μm. More than 100 cells are examined in each sample. c Immunoblot analysis of cell fractionation in TRIM66-KO ESCs overexpressed with HA-tagged WT TRIM66 at different time points after treatment with EPI. d Representative immunofluorescence images of H3K56ac and γ-H2AX foci in WT and TRIM66-KO ESCs, and the intensity profile of both H3K56ac and γ-H2AX across the white line as shown in the images. Scale bar, 10 μm. e Quantification of the colocalized H3K56ac and γ-H2AX foci in WT and TRIM66-KO ESCs. More than 100 cells are examined in each sample. f, g Representative immunofluorescence images and quantification of colocalized Rad51/CtIP and γ-H2AX foci in WT and TRIM66-KO ESCs at 1 h after treatment with EPI. Scale bar, 10 μm. Arrowheads indicates colocalized foci. More than 100 cells are examined in each sample. h Immunoblot analysis of the H3K56ac in WT, TRIM66-KO ESCs, and the TRIM66-KO ESCs overexpressed control plasmid, HA-tagged WT TRIM66, the N1123A or D986A mutant of TRIM66. i Quantification of colocalized H3K56ac and γ-H2AX foci in ESCs described in h. More than 200 cells are examined in each sample. j Quantification of the colocalized Rad51/CtIP and γ-H2AX foci in ESCs described in h at 1 h after treatment with EPI. More than 100 cells are examined in each sample. *represents a difference from WT ESCs, ^#indicates a difference from TRIM66-KO + Ctrl. Data are presented as the means ± SEM. Statistical significance is determined by two-tailed Student’s t-test. *^#p < 0.05, **p < 0.01, ***^###p < 0.001, N.S., no significance. Source data are provided as a Source Data file