Fig. 4. Nrf2 pathway is predominantly activated in EpCAM^high cells and EpCAM knockdown inactivates the Nrf2-ARE pathway.

a. EpCAM^high and EpCAM^low cells were determined in HNSCC patient tumor cells, SCC15 and FaDu cells using flow cytometry. b. Total cellular protein levels of EpCAM, Nrf2, and SOD1 were determined in EpCAM^high and EpCAM^low cells by western blot analysis. c. Nrf2 transcript levels in EpCAM^high and EpCAM^low cells. *p < 0.05 compared to EpCAM^low group. d SOD1, NQO1, and AKRC1 transcript levels in EpCAM^high and EpCAM^lowcells. **p < 0.01 compared to EpCAM^low group. e FaDu cells were transfected with siEpCAM and scrambled siRNA and EpCAM, Nrf2, SOD1, and Sox2 transcript levels were determined by qRT-PCR analysis in EpCAM^high cells. f, g Protein levels of EpCAM, Nrf2, SOD1, and Sox2 were determined by western blot analysis in FaDu and SCC15 HNSCC cells after silencing EpCAM in EpCAM^high cells. h A Luciferase assay was used to detect reporter gene activity from AREs. i Immunostaining of HNSCC cells stained with EpCAM (green), Nrf2 (red), and DAPI (blue) after EpCAM knockdown. Scale bar: 100 μm.