Fig. 4.

PDIA6 inhibits cisplatin-induced autophagy, which contributes to apoptosis suppression in NSCLC cells. (a) Autophagy was evaluated using TEM in NCI-H520/Anip973 control cells or PDIA6 knockdown cells treated with 16 μM cisplatin for 24 h. The arrows indicate autophagosomes or autolysosomes. Scale bar = 2 μm (upper) or Scale bar = 500 nm (lower). (b) NCI-H520/Anip973 control cells or PDIA6 knockdown cells transiently transfected with GFP-LC3II plasmid were treated with 16 μM cisplatin for 24 h. The distribution of GFP-LC3II was visualized by confocal microscopy (upper) and the average number of GFP-LC3 dots in per cell was quantified (lower). Data is expressed as the mean ± SD (n = 3, ***p < 0.001 by Student's t-tests). Scale bar = 25 μm. (c, d) Western blot showing levels of autophagy-related proteins in PDIA6 knockdown (c) or overexpression (d) cells treated with 16 μM (NCI-H520 and Anip973) or 13 μM (A549) cisplatin for 24 h, respectively. (e) NCI-H520/Anip973 control cells or PDIA6 knockdown cells, pretreated with or without 5 mM 3-MA for 2 h, were incubated with 16 μM cisplatin for 24 h. Apoptotic rate was then measured using annexin V/PI double staining. Data is presented as the mean ± SD (n = 3, *p < 0.05, **p < 0.01 and ***p < 0.001 by one-way ANOVA). (f, g) NCI-H520 control cells or PDIA6 knockdown cells, pretreated with or without 5 mM 3-MA for 2 h (f), or transiently transfected with siRNA control (siControl) or siRNA targeting Atg5 (siAtg5) (g), were incubated with 16 μM cisplatin for 24 h. Western blotting was then performed to measure the indicated protein levels.