Fig 2: PCAF, BRD4 determined by Western blot in anti-ISX immunoprecipitates of tumor tissues from patients with liver cancer

Fig 3: SIRT7 augments PCAF binding to MDM2.a Stable SIRT7 knockdown (shSIRT7) or control (shCtr) HCT116 cells were transfected with FLAG-PCAF and treated with 10 µM MG132 for 8 h. Cell lysates were subjected to immunoprecipitation with FLAG-conjugated M2 beads before immunoblotting. b HCT116 cells were transfected with the indicated SIRT7 plasmids or an empty vector and treated with 10 µM MG132 for 8 h. Cell lysates were subjected to immunoprecipitation with an anti-MDM2 antibody before immunoblotting. c HCT116 cells were transfected with SIRT7 (si-SIRT7) or negative control (NC) siRNAs and transfected with FLAG-PCAF before being subjected to glucose deprivation (GD) and 10 µM MG132 treated for 12 h. Total cell lysates were immunoprecipitated with FLAG-conjugated M2 beads before immunoblotting. d HCT116 cells were transfected with the indicated FLAG-PCAF plasmids or an empty vector and treated with 10 µM MG132 for 8 h before being subjected to 10 µM MG132 treated with or without glucose deprivation (GD) for 12 h. Total cell lysates were immunoprecipitated with FLAG-conjugated M2 beads before immunoblotting. e HCT116 cells were transfected with the indicated FLAG-PCAF and Myc-Ub plasmids and treated with 10 µM MG132 for 8 h. Total cell lysates were immunoprecipitated with an anti-MDM2 antibody and the ubiquitination levels were analyzed by immunoblotting. f PCAF(WT) and PCAF (KO) HCT116 cells were transfected with the indicated plasmids and then treated with CHX for the indicated time. Cells were harvested and MDM2 protein levels were measured by immunoblotting. g Quantitation of MDM2 protein levels. Immunoblots in f were scanned and normalized to ß-actin. The data represent the means ± SD (n = 3 experiments).

Fig 4: Effects of GNAT siRNA treatment on UCC clonogenicity. Colony formation assays after treatment of the indicated UCCs with siRNAs against GCN5, PCAF (A) or both HATs (B). Irrelevant designates treatment with non-targeting siRNA. Representative plates from triplicate experiments are shown.

Fig 5: Reduced levels of mature B lymphocytes in spleens and blood of 8–12-week-old mice of indicated genotypes. (A) Proportions of B220+IgM+ mature B cells in spleen. WT vs. Pcaf-/-, n.s. p = 0.4777; WT vs. Gcn5-/-, ns, p = 0.2532; WT vs. Gcn5-/-Pcaf-/-, **** p < 0.0001; Pcaf-/- vs. Gcn5-/-, * p = 0.0119; Pcaf-/- vs. Gcn5-/-Pcaf-/-, **** p < 0.0001; Gcn5-/- vs. Gcn5-/-Pcaf-/-, *** p < 0.0001. (B) Summary of experiments as shown in (A), number of cells. WT vs. Pcaf-/-, n.s., p = 0.9212; WT vs. Gcn5-/-, * p = 0.0214; WT vs. Gcn5-/-Pcaf-/-, **** p < 0.0001; Pcaf-/- vs. Gcn5-/-, ** p = 0.0053; Pcaf-/- vs. Gcn5-/-Pcaf-/-, **** p < 0.0001; Gcn5-/- vs. Gcn5-/-Pcaf-/-, * p < 0.0269. (C) Proportions of B220+ B cells and CD3+ T cells in blood. (D) Summary of several experiments from (C) reflecting proportions of B cells in blood. WT vs. Pcaf-/-, n.s. p = 0.9196; WT vs. Gcn5-/-, n.s., p = 0.1204; WT vs. Gcn5-/-Pcaf-/-, **** p < 0.0001; Pcaf-/- vs. Gcn5-/-, n.s., p = 0.5398; Pcaf-/- vs. Gcn5-/-Pcaf-/-, **** p < 0.0001; Gcn5-/- vs. Gcn5-/-Pcaf-/-, *** p < 0.0001. Gcn5-/- and Gcn5-/-Pcaf-/- indicate Cd19Cre-dependent inactivation of Gcn5 in B-cell lineages.