Extended Data Fig. 7: TRMT6 knockdown restricted histone synthesis.

(a) The protein levels of newly synthesized cytoplasmic histones (H1, H2B, H3, and H4) in CRC cell lines upon thymidine incubation and removal. Experimental setup (top panel), #1: The cells were incubated with medium for 24 h; #2: The cells were incubated with both medium and thymidine for 24 h respectively; #3: Based on #2, thymidine was removed, and the cells were further cultured in the medium for another 24 h. In the bottom panel, WB analysis showing that cytoplasm-extracted histones were decreased upon thymidine treatment and returned to normal levels after thymidine removal. Thymidine: an inhibitor of DNA synthesis. (b) WB was performed to confirm the efficiency of cytoplasmic and nuclear protein separation, using α-tubulin as a cytoplasmic protein control and Lamin A/C as a nuclear protein control. (c) In TRMT6-depleted and control cells, cytoplasm-extracted and acid-extracted proteins were analyzed by western blot and Coomassie staining (12.5% SDS–PAGE). Upon TRMT6 knockdown, the levels of newly synthesized cytoplasmic histones (cytoplasm-extracted) were decreased, while the levels of chromatin-bound histones (acid-extracted) remained unchanged. (d) The experimental design is as depicted in the diagram: following transfection, cells were incubated with thymidine nucleoside for 20 h, after which the thymidine nucleoside was removed, and the cells were further cultured in complete DMEM medium until the specified time point. The experiments in a, b, and c were independently performed three times, all with similar results.