Figure 4.

CTN06 induces autophagy as well as apoptosis in prostate cancer cells. (a) Growth inhibition of CTN06 to LNCaP, Du145, PC3 prostate cancer and normal prostate (RWPE1) cells. Cells were seeded at 5000 cells/well in 96-well plate overnight and treated with CTN06 at the indicated concentrations. The cell viability was measured using MTT assay after 72 h. Columns, mean; bars, standard deviation, n=3. Inhibition of Btk by CTN06 (0.5 μM) in those cells was measured using western blot. (b) Induction of autophagy in PC3 cells by CTN06. PC3 cells were stably transfected with GFP-LC3 and were grown in 6-well plate to 50% confluence and treated with CTN06. Autophagy was visualized by GFP-LC3 ‘puncta' at 2 and 24 h and immunoblot of endogenous LC3 isoforms 24 h after treatment. (c) Induction of apoptosis of PC3 cells following treatment with CTN06. PC3 cells were seeded at 10⁶ cells/ml (2 ml) in a 6-well plate overnight and then treated with CTN06 at the indicated concentrations for 24 h. Apoptosis was analyzed using PI staining as well as Annexin V-FITC apoptosis detection kit. Columns, mean; bars, standard deviation, n=3. In all, 0.5 and 2 μM are significantly different from 0 μM (*P<0.05, one-way ANOVA with Tukey test for pairwise comparison)