Fig. 4
Cross-resistance to smoke and cisplatin is mediated by Nrf2. A Using a 72-h Hoechst assay we compared the cell number following cisplatin (left upper panel) or smoke exposure (right upper panel) in the parental HN30 cell line and its cisplatin-resistant derivative HN30R8. We then compared cell number following smoke exposure (left bottom panel) or cisplatin (right lower panel) in the parental HN30 cell line and its chronically smoke-exposed derivative HN30 3%. B HN30 cells were stably infected with either control shRNA or KEAP1 shRNA resulting in increased Nrf2, Gpx2 and Nqo1 levels when Keap 1 levels were reduced. C HN30 KEAP1 shRNA cells demonstrated increased resistance to both cisplatin and smoke exposure (Hoechst, 72 h). D KEAP1 wild-type (wt) and KEAP1fl/fl clones A, D, E, and F generated from HNSCC GEMM models were harvested for Nrf-2 and Gpx2 protein measurements. E KEAP1fl/fl clones E and F demonstrated increased resistance to both cisplatin and smoke compared to the KEAP1 wildtype cells (Hoechst, 72 h). F HN30 cells were stably infected with either human NRF2 overexpression (OE) plasmid or empty vector (EV). Whole cell lysates and cell fractions were analyzed for Nrf2, Keap1, Gpx2, Nqo1 and p65 protein levels. α-Tubulin served as loading control for the cytoplasmic fraction, and histone H3 served as loading control for the nuclear fraction. G HN30 cells with NRF2 OE demonstrated increased resistance to both cisplatin and smoke (Hoechst, 72 h). Hoechst data are shown as means, normalized to control condition; error bars indicate standard deviation; p values are denoted as *p < 0.05, **p < 0.01, and ***p < 0.001