Supplementary Figure Legend

FigureS1 Depletion of p300, CBP or P/CAF increases the population of cells arrested at mitosis.

(a) HeLa cells were transfected with luciferase shRNA, p300 shRNA, CBP shRNA, and P/CAF shRNA. 48 hr after transfection, the cells were harvested and analyzed by flow cytometry. (b) HeLa cells were infected with a recombinant adenovirus expressing p300 shRNA (rAd-p300 shRNA), CBP shRNA (rAd-CBP shRNA), P/CAF shRNA (rAd-P/CAF shRNA), or GFP (rAd-GFP) as a control. Infected HeLa cells were then analyzed by flow cytometry.

Figure S2HeLa cells with depletedp300, CBP or P/CAF showed augmented chromosome aneuploidy.

HeLa Con, p300KD, CBPKD, and P/CAFKD cells werecultured, stained with propidium iodide, and then analyzed by flow cytometry to evaluate their DNA contents.

Figure S3 Depletion of p300 interferes with the normal timing of mitotic progression in human primary amniocytes (AF9-1).

(a) Immunofluorescence analysis of AF9-1 cells following taxol treatment. Cells werefixed with 4% paraformaldehyde and then stained with anti-αTubulin (green), anti-phospho-H3 (red) antibodies and DAPI (blue). (b) Histogram showing the percentage of mitotic cells following taxol treatment. Approximately 1000 cells were scored by immunofluorescence for each time point. Data represent the average number of cells positive for phospho-H3 from three independent experiments; approximately1000 cells/sample were counted in each experiment. (c) AF9-1 cells were infected with a recombinant adenovirus expressing p300 shRNA (rAd-p300 shRNA) and luciferase shRNA (rAd-Luc shRNA) as a control, and then cultured in the absence (Asyn) or presence of taxol for 24 hr (T24) and 48 hr (T48). Infected AF9-1 cells were analyzed by flow cytometry.

Figure S4 HAT activity was maximal in mitotic cells, but was uncoupled from mitotic chromosome condensation

(a) HeLa cells were synchronized by treatment with nocodazole (100 ng/ml) for 16 hr and then released from nocodazole treatment to generate a population of post-mitotic cells, as indicated. Asynchronous (Asyn) and synchronized HeLa cells were harvested, stained with propidium iodide, and then analyzed by flow cytometry to determine their DNA content (left panel). HAT activity in the nuclear fractions of asynchronous and synchronized HeLa cells was determined as described in the Materials and Methods section (right panel). Values shown are the means ± SD of triplicate measurements. (b) HeLa cells were synchronized by double thymidine block (Thy-DB) and release. Cells were stained with propidium iodide and then analyzed by flow cytometry at the indicated times (left panel). Ponceau-stained core histones (H2A, H2B, H3 and H4) represent loading controls. Nuclear extracts (containing chromatin) were analyzed by immunoblotting using antibodies against phospho-H3 (P-H3), H3 Lys 9-acetyl (H3AcK9), H3 Lys 14-acetyl (H3AcK14) (Upstate Biotechnology) or actin (Sigma) (right panel). (c) HeLa cells were co-immunostained with anti-acetyl lysine (AcK, shown in green) and anti-pericentrin (Abcam; shown in red), and DNA was visualized with Hoechst dye (shown in blue). G1/S and G2 cells were obtained by synchronization with Thy-DB and etoposide treatment, respectively. NEBD indicates nuclear envelope membrane breakdown. (d) HeLa cells were transfected with either a control shRNA or p300 shRNA and then cultured for an additional 36 hr. Transfected cells were stained with anti-tubulin (Sigma) and anti-acetyl lysine (AcK, Cell Signaling technology) and Hoechst dye.