Figure 4.

Knockdown of p300ΔC reduces proliferation of SUDHL2 cells and increases A20 and IκBα expression in RC-K8 cells. (a) Anti-p300 Western blotting was performed on whole-cell extracts from SUDHL2 cells expressing p300 shRNA or control shRNA. Anti-β-tubulin Western blotting is a loading control. (b) 10⁵ SUDHL2 cells expressing p300 shRNA or control shRNA were plated in 500 μl RPMI/10% FBS in 16-mm wells. On each day, three wells of each cell type were counted. (c) Soft agar colony assays were performed using SUDHL2 cells expressing p300 shRNA or control shRNA. Results are averages of three experiments performed with triplicate plates containing 2000 or 5000 cells. Colony numbers are normalized to the number of colonies formed by SUDHL2 cells expressing control shRNA (100). Error bars represent standard deviation. (d) Reverse transcribed total RNA from RC-K8 cells expressing p300 shRNA or a control shRNA was subjected to real-time qPCR using gene-specific primers. mRNA expression values from RC-K8 cells expressing p300 shRNA were compared to values from RC-K8 cells expressing control shRNA to determine the fold change in expression. Error bars represent standard error of the mean. Asterisks indicate p-values < 0.05 for the difference in mRNA levels relative to control cells (=1.0) using a two-tailed t test. (e) Anti-p300, anti-A20, and anti-IκBα Western blotting was performed on whole-cell extracts from RC-K8 cells expressing p300 shRNA or control shRNA. Anti-β-tubulin Western blotting is a loading control. (f) RC-K8 cells were subjected to crosslinking with formaldehyde and extracts from isolated nuclei were then immunoprecipitated using normal rabbit IgG or anti-p300 antiserum. The crosslinks were reversed and DNA in immunoprecipitates was subjected to qPCR using primers spanning the A20 promoter region. Relative A20 promoter PCR product was normalized to the IgG control (1.0). Error bars represent the standard error of the mean.