Fig. 3.
Pak2 is required for de novo H3.3 deposition in senescence cells. (A and B) H3.3 occupancy at the (A) p16INK4a transcription start site (TSS) and (B) IL-6 enhancer region was determined by ChIP-qPCR (n = 3, mean ± SEM, *P < 0.05, **P < 0.01). Values were normalized to input. (C) Experimental scheme for in vivo quench–chase–pulse (QCP)-SNAP labeling of de novo assembly of H3.3 in senescent IMR90 cells overexpressing H3.3-SNAP. (D) Western blotting analysis of lysates from IMR90 cells expressing H3.3-SNAP treated in C. Membranes were probed for the indicated antibodies. α-Tubulin was used as a loading control. (E and F) Pak2 and HIRA depletion compromised the deposition of newly synthesized H3.3 in senescent cells. Pak2 was depleted in H3.3-SNAP-tagged IMR90 cells. After old H3.3-SNAP was blocked using a nonfluorescent blocker, new H3.3-SNAP was labeled with TMR-STAR (the red fluorescent substrate for the SNAP tag) and visualized using fluorescence microscopy (E). The SNAP-TMR signal intensity was quantified and reported as mean ± SEM of 3 experiments (F, **P < 0.01). Depletion of HIRA was used as a control.