Research Paper Volume 16, Issue 3 pp 2789—2811

Figure 5. Targeting ATF4 increases the sensitivity of PDT by inhibiting autophagy. HOS cells in MPPα-PDT+GSK2656157 group were pretreated with 5 mM GSK2656157 for 1 h before MPPα-PDT treatment. HOS cells were transfected with siRNAs and then treated with MPPα-PDT. (A) Protein ATF4 or mRNA ATF4 was detected by western blot and represents the transfection efficiency. (B) Cell viabilities were detected by CCK-8 after treated with SiRNA-ATF4 (Si-ATF4), SiRNA-negative control (Si-NC), MPPα-PDT and group MPPα-PDT combined with Si-ATF4. (C) Following indicated treatments, cells were harvested and ATF4, p21, PARP and cleaved caspase-3 levels determined by western blot. (D, E) Apoptotic rate or JC-1 stain was examined by flow cytometry or fluorescence microscope (×200). (F) Following indicated treatments, cells were harvested and LC3, P62, Atg5 and Beclin1 levels were determined by western blot. (G) Adenovirus-GFP-LC3 was transfected into the HOS cells after treated, the LC3 fluorescent particles were observed by laser confocal microscope (×400). ^*P < 0.05 vs. control group, data are shown as mean ± SD of 3 independent experiments.