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. 2019 Mar 25;42:311–325. doi: 10.1016/j.ebiom.2019.03.045

Fig. 5.

Fig. 5

PDIA6 inhibits cisplatin-induced apoptosis and autophagy through JNK/c-Jun signaling pathway. (a) Human phospho-kinase array profile of differential phospho-proteins between NCI-H520 control cells and PDIA6 knockdown cells treated with 16 μM cisplatin for 24 h. Each phospho-protein is represented in duplicate. Red and blue boxes indicate noticeably altered c-Jun and JNK phosphorylation levels, respectively. (b) Western blotting analysis of the impact of PDIA6 knockdown on the activity of JNK and c-Jun in NCI-H520 and Anip973 cells treated with 16 μM cisplatin for 24 h. (c) pGMLV-PA6-PDIA6 or control vector together with pAP-1-Luc reporter or pGM-Luc control reporter were co-transfected to A549 cells with pRL-TK Renilla. After 24 h, cells were incubated with 13 μM cisplatin for another 24 h. Firefly luciferase activity was then detected and normalized to the Renilla luciferase activity. Data is expressed as the mean ± SD (n = 3, ***p < 0.001 by Student's t-tests). (d) NCI-H520 control cells or PDIA6 knockdown cells, pretreated with or without 20 μM SP600125 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 expressed as the mean ± SD (n = 3, *p < 0.05 and ***p < 0.001 by one-way ANOVA). (e) NCI-H520 control cells or PDIA6 knockdown cells transiently transfected with GFP-mRFP-LC3 plasmid were pretreated with or without 20 μM SP600125 for 2 h. After that, cells were incubated with 16 μM cisplatin for 24 h. Images were then acquired by confocal microscopy (left) and the average number of yellow dots (autophagosomes) or red-only dots (autolysosomes) in the merged images of per cell was quantified (right). Data is expressed as the mean ± SD (n = 3, ***p < 0.001 by one-way ANOVA). Scale bar = 25 μm. (f, g) NCI-H520 control cells or PDIA6 knockdown cells, pretreated with or without 20 μM SP600125 for 2 h (f), or transiently transfected with the siRNA control (siControl) or siRNA targeting JNK (siJNK) (g), were incubated with 16 μM cisplatin for 24 h. Western blotting was then performed to measure the expression of cleaved caspase-3, total JNK, phospho-JNK (p-JNK), phospho-c-Jun (p-c-Jun), and LC3I/II. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)