Fig. 6

Proteasome activity was required for MTX-induced apoptosis. A. Proteasomes activation assay. LNCaP and 22RV1 cells were treated with vehicle, MTX (1 μM) for 24 h. Proteasome activation of LNCaP (up) and 22RV1(down) treated with indicated concentrations were indicated as hydrolysis rate succinyl-Leu-Leu-Val-Tyr-amc (Suc-LLVY-amc). B-C. LNCaP cells were treated with vehicle, BZM (200 nM), MTX (1 μM) alone and combination (BZM, 100 nM; MTX, 0.5 μM) for 24 h. B. Representative flow cytometry plots. C. Quantitative data of apoptosis analysis. Early apoptotic was defined as Annexin positive population, later apoptotic as Annexin/PI positive. D. Cells proliferation was determined by CCK-8. Data are presented from three independent experiments. All data are presented as mean ± s.e.m. The asterisk indicates a significant difference. (One-way ANOVA, *p < 0.05; **p < 0.01)