Enhanced p62-NRF2 Feedback Loop due to Impaired Autophagic Flux Contributes to Arsenic-Induced Malignant Transformation of Human Keratinocytes

Figure 3

Amplification of p62-NRF2 feedback loop is required for the acquisition of arsenite-induced malignant phenotypes. (a) mRNA levels of p62 in HaCaT cells infected with lentiviral vector expressing shRNA targeting p62 (p62-KD) or scrambled nontarget negative control (SCR). (b) Protein levels of p62 and NRF2 in p62-KD and SCR cells. Left: representative image; right: quantification of protein levels of p62 and NRF2 determined with Western blot. (c) mRNA levels of NRF2 downstream genes, AKR1C1, GCLC, and NQO1, in p62-KD and SCR cells. (d) mRNA levels of NRF2 and its downstream genes in chronic arsenite-exposed cells with NRF2 knockdown (NRF2-KD). (e) mRNA and protein levels of p62 in NRF2-KD cells analyzed with RT-PCR (left) and Western blot (right), respectively. Upper right: representative image for Western blot; lower right: quantification of p62 protein levels determined with Western blot. (f) Invasion capacity determined by xCELLigence RTCA. Cell index at 45 h after seeding was used to assess invasion capacity. (g) Colony formation in soft agar. Representative image (upper) and quantification of the colonies (lower). Scale bar is 100 μm. As (As+): cells were chronically exposed to 100 nM of sodium arsenite for 30 weeks. Con: passage-matched nontreated cells. except for colony formation assay, in which . , compared with control (As-) compartment. ^#, compared with SCR (NRF2-KD- or p62-KD-) compartment.

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