Fig. 6.

Therapy-induced senescence reduces readthrough in cancer cells. (A) SA-β-gal staining of PC-3 cells treated 24 h with vehicle and/or 100 nM of camptothecin and fixed 7 days later (day 7), the percent positive cells ±SD is shown in lower corner, n=3. (B) Immunoblots in PC-3 cells transduced with luciferase reporter (AQP4, UGA) and treated as in A for the proteins: RB(pS795) [phosphorylated RB on serine 795], MCM6, H3(pS10) [phosphorylated H3 on serine 10] and tubulin. Blots are representative of three independent experiments with similar results. (C) Normalized Fluc/Rluc ratios of indicated readthrough reporters in cells as in A. Normalizations are presented as means relative to vehicle treated cells from (UGA) n=4 and (AQP4) n=5 independent experiments each with technical triplicates. (D) Normalized Fluc/Rluc ratios in of MDA.MB.231 cells transduced with the luciferase reporters (UGA, AQP4) and treated as in A. Normalizations are presented as means relative to vehicle treated cells, n=3. (E) Immunoblots for phosphorylated RB at serine 795 [RB(pS795)], MCM6 and tubulin in cells as in D, n=3. (F) Normalized Fluc/Rluc ratios of indicated readthrough reporters in PC-3 cells as in A, 21 days post-treatment when the cell population bypassed senescence. For all luciferase assays, n=3, error bars indicate SD of technical triplicates of each experiment. *=P<0.05, **=P<0.01, ***=P<0.001 using two-tailed Student's t-test. (G) Immunoblots for phosphorylated RB at serine 795 [RB(pS795)] and tubulin in cells as in E, n=3.