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. 2021 Mar 11;11(11):5232–5247. doi: 10.7150/thno.53417

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NRF2 promotes malignant features of HNSCC in vitro. Three human HNSCC cell lines, HONE-1, Ca9-22-D1 and OECM-1, were transiently transfected with NRF2 siRNA (siNRF2 #1 and siNRF2 #2) or scramble control (siControl). The motility and invasiveness of the transfected cells were evaluated by trans-well migration (A) and invasion (B) assays. The cells that had migrated or invaded were quantified and expressed as a percentage relative to siControl group. (C) The effects of NRF2-knockdown on cell growth of HNSCC cells. Cell number was determined using the methylene blue assay. (D) Migration ability was determined in DOK cells stably overexpressing NRF2 that were treated with NRF2 siRNA or a non-targeted control. To present relative cell migration, the value from the vector-only control DOK cells (blue bar) was set at 100%. (E) The growth rates of DOK cells stably overexpressing NRF2 and treated with NRF2 siRNA or a non-targeted control under low serum conditions were measured. The data were expressed as the percentage relative to the vector-only control DOK cell (MOCK) which was treated with negative control siRNA (blue bar). (F) Alteration of cellular properties with long-term exposure to nicotine or arecoline in dysplastic DOK cells. The nicotine- and arecoline-transformed DOK cells were established from DOK cells treated with non-toxic concentrations of carcinogens (nicotine: 500 µM; arecoline: 50 µM) for 1 year. The levels of NRF2, KEAP1, phospho-EGFR (Tyr1068), EGFR, B-RAF, and c-MYC in total lysates were assessed using Western blot analysis. The cell proliferation rates were measured by methylene blue assay after 72 h incubation and calculated as the fold increase compared to proliferation of the parental DOK cells (black bar). Top: representative Western blots. Bottom: the cell proliferation index. The effects of knockdown of MYC on NRF2 protein level in HNSCC cells (G) and carcinogen-transformed DOK cells (H). (I) RT-qPCR analysis of NRF2 mRNA levels in MYC-knockdown OEC-M1 cells. (J) ChIP-qPCR analysis of c-MYC binding at NRF2 promoter in MYC-knockdown OEC-M1 cells. All data are expressed as the mean ± S.D. from three individual experiments. * p < 0.05; ** p < 0.01; *** p < 0.001. vs. control.