Figure 2.

SAHA induces p21^WAF1 mRNA and protein in T24 cells. T24 cells were cultured with SAHA (7.5 μM) for the indicated times. (A) Western blot analysis of p21^WAF1 protein expression. Protein extracts were prepared and resolved (25 μg) on 15% SDS/PAGE. p21^WAF1 protein was detected by using the mouse monoclonal anti-p21^WAF1 F5 antibody. A parallel gel was stained with Coomassie blue for a loading control. (B) Northern blot analysis of p21^WAF1 mRNA expression. Total RNA (10 μg) was isolated and fractionated on 1.2% agarose and transferred to a nylon membrane. The membrane was probed by using ³²P-random-labeled human p21^WAF1 cDNA, the γ-actin cDNA, and a 50-mer oligonucleotide specific for 18S rRNA. (C) Run-on transcription assay by using nuclei from SAHA-induced T24 cells. Nuclei were prepared and labeled with [α³²P]-CTP. Equal levels of radioactivity (approximately 5 × 10⁷ cpm) were incubated with filters containing 5 μg of the indicated plasmid cDNA. Radioactivity bound to the filters was quantified and normalized to actin at each time point examined. The actin-normalized value for the untreated sample was adjusted to a value of 1 to determine fold increase after culture with SAHA.