Fig. 7. IL-6 (Interleukin-6) and p62 are involved in the activation of the Nrf2 pathway and cell death resistance in EpCAM^high cells.

a Real-time PCR analysis of IL-6 expression in HNSCC tumor tissues from matched adjacent normal (n = 10), untreated (surgery only; n = 10), cisplatin treated (n = 10), doxorubicin (n = 10), and chemo-radiotherapy (n = 10) treated tumor tissues. Transcripts levels were normalized to beta-actin. b The immunofluorescence images of cytoplasmic and nuclear Nrf2 in FaDu cells after 5-day cisplatin treatment with or without 150 pg/mL IL-6 (legend: 1-cytoplasmic; and, 2-nuclear Nrf2; Scale bar = 100 μm). c Nrf2 protein levels after 5-day post-treatment with cisplatin or combination of cisplatin and 150 pg/mL of IL-6, analyzed by western blotting. d Keap1 mRNA and protein expression in FaDu cells 5-day post-treatment with cisplatin alone or with 150 pg/ml of IL-6. e ROS level was determined in FaDu and SCC15 cells after treating cells with cisplatin, IL-6 and combination of IL-6 and cisplatin. f After 72 h cisplatin treatment with vehicle or 150 pg/mL IL-6 or combination of IL-6 and 30 ng/mL tocilizumab, cell lysates were subjected to western blotting and the levels of SOD1 and Nrf2 proteins were determined. g After 72 h cisplatin treatment with vehicle or 150 pg/mL IL-6 or IL-6 with 30 ng/mL tocilizumab, the ROS production was analyzed. h p62 and LC3B were measured in EpCAM^low and EpCAM^high FaDu cells by western blotting. i p62 protein was determined in EpCAM^low and EpCAM^high FaDu cells following scrambled siRNA and EpCAM-siRNA transfection. Quantification and representative images of spheres formed by si-scrambled and EpCAM-siRNA transfected EpCAM^high cells are presented. Scale bar 100 μm. Values represent three separate experiments. **p < 0.01 compared with si-scramble group. j EpCAM^high cells were transfected with scrambled or p62-siRNA and protein levels of Nrf2, p62, Keap1, SOD1, EpCAM, and LC3B were assessed. Quantification analysis and representative images of spheres formed by si-scrambled and p62-siRNA transfected EpCAM^high cells are presented. Scale bar 100 μm. Values represent three separate experiments. **p < 0.01 compared with si-control group. k, l Transcript levels of EpCAM, LC3B, SOD1, HO-1, and AKRC1 were determined in si-scrambled and p62-siRNA transfected cells by qRT-PCR. Values represent mean ± SD from three separate experiments. *p < 0.05; **p < 0.01. Values compared with si-control group.