Figure 4.

BMP7 induces reversible senescence. (A) Effect of BMP7 on the growth of PC3 mm cells was measured by the MTT assay. Cells were treated with 200 ng/ml BMP7 continuously (green), with BMP7 between day 4 and 11 followed by BMP7 withdrawal (red), or with control vehicle (black; n = 5). (B) PC3 mm cells were cultured with 10 ng/ml EGF or 200 ng/ml BMP7 alone, or EGF with BMP7, or EGF with BMP7 followed by BMP7 withdrawal, and the expression of p-Erk, Erk, p-p38, p38, and β-tubulin was examined by Western blot. (C) Effect of BMP7 on growth arrest was examined by SA–β-gal staining (top) and cell cycle analysis (bottom). PC3 mm cells (left) and C4-2B cells (right) were cultured with (+/+) or without (−/−) BMP7 for 96 h. +/− cells were treated with BMP7 for 48 h followed by withdrawal of BMP7 and further incubation for 48 h (n = 3). (D) Effect of BMP7 on senescence was examined by SA–β-gal staining. DU145, LNCaP, C4, and ALVA were cultured with (+/+) or without (−/−) BMP7 for 96 h. +/− cells were treated with BMP7 for 48 h followed by withdrawal of BMP7 and further incubation for 48 h (n = 3). (C and D) The insets show representative images of three independent experiments. Bars, 50 µm. (E and F) PC3 mm cells that had either scrambled shRNA (scramble) or shRNA for NDRG1 (shNDRG1) were cultured with BMP7 or control vehicle, and the knockdown of NDRG1 was confirmed by qRT-PCR (n = 3; E). These cells were stained for SA–β-gal activity (n = 4; F). (G) Effect of NDRG1 on senescence was examined by SA–β-gal staining (bottom). PC3 mm cells stably expressing Tet-NDRG1 were cultured with (+/+) or without (−/−) tetracycline for 96- or 48-h treatment followed by 48-h withdrawal of tetracycline (+/−; n = 4). The expression of NDRG1 and β-tubulin was examined by Western blot (top). *, P < 0.05; **, P < 0.01; ***, P < 0.001. The experiment in A was performed twice, experiments in B–G were performed three times independently, and representative data are shown. Results are shown as mean ± SEM.