Fig. 7: The effect of activated AR on CCND1 expression by changing histone modifications of its promoter. | Cell Death & Disease

Fig. 7: The effect of activated AR on CCND1 expression by changing histone modifications of its promoter.

From: The beneficial androgenic action of steroidal aromatase inactivators in estrogen-dependent breast cancer after failure of nonsteroidal drugs

Fig. 7

Cell culture method and treatment was as shown in Fig. 2d. The experimental protocol was as described in “Materials and methods”. a and b The expression of the cyclin D1 protein. Blots were also probed for tubulin (bottom) to verify equal amounts of protein loaded in each lane. The densitometric evaluation is calculated by ImageJ. c The abundance of cyclin D1 mRNA has been detected by real-time reverse transcription-PCR, as described in “Materials and methods”. dh MCF-7 cells were grown in 10 cm dishes. Confluent cultures (80%) were shifted to PRF for 24 h and then treated with indicated compounds at a concentration of 10−7 M or left untreated in PRF-CT for 2 h. ChIPs were carried out on serum starved MCF-7 cells as described in “Materials and methods”. Cells were lysed and proteins were precipitated using antibodies against AR (d), H3K4me3 (e), H3K9me3 (f), H3K9ac (g), H3K27ac (h) or rabbit IgG (2 μg/sample each). In control samples (Ig), normal rabbit IgG was used instead of the primary antibodies as control of antibody specificity. Inputs DNA were amplified as loading controls. Five regions of CCND1 promoter were examined by quantitative PCR analysis. The distance from TSS of the CCND1 promoter portions is indicated in Table 1. The region containing the ARE site was detected by PCR with specific primers (p1-ARE) for antibodies of AR and H3K4me3. Specific primers P4 and p5 were used to investigate the changes in methylation or acetylation of H3K9. H3K27 acetylation does not change as judged by using all pairs of primers. These data are representative of three separate experiments, each in triplicate; bars, SEM. *P ≤ 0.05 vs. control

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