Fig. 3: p21 inhibition induces autophagy flux.
A HCT116 p21wt and null cells were incubated with 5 µM CQ for 24 h. Cell lysates were processed for western blot analysis with indicated antibodies. Relative LC3-II expression level among experimental groups were quantified from cell lysates of three independent experiments and graphically represented as mean ± SE. B Analysis of LC3 by immunoblotting in HCT116 p21+/+ cells treated with vehicle or UC2288 in the presence or absence of 5 µM CQ (2 h pre-incubation). Relative LC3-II expression level among experimental groups were quantified from cell lysates of three independent experiments and graphically represented as mean ± SE. C HCT116 p21+/+ cells were transiently transduced for 24 h with control or p21-specific shRNAs and incubated with or without 5 µM CQ for an additional 24 h before being analyzed for LC3 conversion by western blot assay. Relative LC3-II expression level among experimental groups was quantified from cell lysates of three independent experiments and graphically represented as mean ± SE. D Representative confocal micrographs of GFP-LC3 expressing HCT116 p21+/+ cells treated with vehicle or UC2288 in the presence or absence of 5 µM CQ (2 h pre-incubation). Rapamycin (2 µM for 24 h incubation) was used as the standard autophagy inducer in this study. E HCT116 p21+/+ cells, with stable expression of GFP-LC3-RFP-LC3ΔG, were treated with UC2288 after 2 h pre-incubation with 5 µM CQ and examined under confocal microscope. F GFP-LC3 expressing HCT116 p21wt cells were incubated with UC2288 alone or in combination with rapamycin. Cells were then analyzed by flow cytometry to measure GFP fluorescence intensity. Data presented as the mean of three (n = 3) independent experiments ± SE. **P < 0.01, ***P < 0.001.
