Research Paper Volume 16, Issue 3 pp 2789—2811

Figure 6. The pro-survival effect of P21 is achieved by regulating autophagy after MPPα-PDT. (A) ChIP analysis was used to detect ATF4 binding to the first intron of p21. HOS cells were transfected with siRNAs and then treated with MPPα-PDT. (B) Protein P21 were detected by western blot to represent the transfection efficiency. (C) Following indicated treatments, cells were harvested and P21 and cleaved caspase-3 levels were determined by western blot. (D, E) Apoptotic rate or JC-1 stain were examined by flow cytometry or fluorescence microscope (×200). (F) Protein P21 was detected by western blot after transfected by lentivirus-overexpress P21. (G) Following indicated treatments, cells were harvested and LC3, P62, Atg5 and Beclin1 levels were determined by western blot. (H) Adenovirus-GFP-LC3 was transfected into the HOS cells after treated, the LC3 fluorescent particles were observed by laser confocal microscope (×400). (I) Protein CHOP, cleaved-caspase3 were detected by western blot. (J) Apoptotic rate was examined by flow cytometry. ^*P < 0.05 vs. control group, data are shown as mean ± SD of 3 independent experiments.