Figure 5.

Cyclin D1 is associated with the p21waf1 promoter to prevent its expression in breast cancer cells (A) MCF-7 cells were serum-starved for 2 d and stimulated for 4 h with IL-6 (20 ng/ml) or 10% serum as indicated. Total RNA was prepared and 20 μg of RNA was subjected to Northern blot analysis using a human cDNA probe (lanes 1–3). The membrane was striped and reprobed with a 18S oligonucleotide (bottom panel). The recruitment of cyclin D1 to the p21waf1 promoter was analyzed in parallel by ChIP after IL-6 stimulation (lanes 4–5). (B) Serum-starved MCF-7 cells were pretreated for 1 h with LY294002 (50 μM, lanes 3 and 4, 7 and 8) or with dimethyl sulfoxide (DMSO; vehicle, lanes 1 and 2, 5 and 6), and then left untreated or stimulated with IL-6 for 20 min. Cyclin D1 expression was analyzed by Western blot (lanes 1–4). ChIP analysis were performed to analyze the recruitment of cyclin D1 on the p21waf1 promoter after LY294002 pretreatment (lanes 5–8). Cells were treated as explained in A and stimulated for 20 min, and ChIP experiments were performed as described Figure 2. (C) Northern blot analysis of the expression of the p21waf1 mRNA in the presence or absence of LY294002. (D) Serum-starved MCF-7 cells were pretreated for 1 h with LY294002 (+) or with DMSO (–) and were then stimulated with IL-6 for 20 min. The association of STAT3 and CBP with the p21waf1 promoter was analyzed by ChIP using one pair of primers that cover the STAT3 binding site (lanes 1–4). The association of the RNA pol II with the p21waf1 promoter was analyzed in parallel with one pair of primers that cover the TATA box binding site (lanes 5–8).