p300 HAT activity is required for Vpr-induced PCS. (A) HAT activity in Flag-p300– or Flag-p300ΔHAT–expressing cells. The acetyltransferase deletion mutant p300ΔHAT (Δ1,472–1,522) displayed no activity in a HAT activity assay. Mean values are shown. ND, none detected. Results shown are representative of two independent experiments. (B) A schematized experimental procedure of C. (C) PCS frequency in Vpr-expressing and -nonexpressing cells transfected with pFlag-p300 or pFlag-p300ΔHAT (48 h). Values represent the mean ± SD for independent experiments (n = ∼2–8). Cont, control. (D) The chromatin loading of Flag-p300 or Flag-p300ΔHAT protein in cells expressing Vpr. Cells were preextracted with PBS containing Triton X-100 and stained for Flag M2 epitope and DNA. Levels of chromatin-bound Flag-p300 or Flag-p300ΔHAT were comparable in Vpr-expressing cells. Non–Vpr-expressing cells contained no chromatin-bound Flag-p300 or Flag-p300ΔHAT. Bar, 10 µm. (E) Quantification of Flag-p300/Flag-p300ΔHAT signal intensities in D. Results shown are representative of two independent experiments. Arb., arbitrary. (F, top) A schematized experimental procedure of G and H. Gray underlined letters represent mutation sites in the RNAi-resistant wt and p300ΔHAT cDNA plasmids. (bottom) Mutations introduced to prepare RNAi-resistant Flag-p300wt (pFlag-p300wtR) and RNAi-resistant p300ΔHAT (p300ΔHAT-R). (G) Western blotting to verify that the expression of pFlag-p300wtR was not affected by p300RNAi. Arrowhead, Flag-EGFP; dot, nonspecific band. βTub, β-tubulin. (H) PCS frequency in Vpr-expressing cells transfected with pFlag-p300wtR or p300ΔHAT-R (48 h) and plasmids carrying RNAi-resistant Flag-p300wt and RNAi-resistant p300ΔHAT. Values represent the mean ± SD of three independent experiments. PCS frequency was significantly lower in cells expressing p300ΔHAT-R than in those expressing p300wtR. *, P = 0.0003.