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

Figure 4

Arsenite inhibits autophagosome-lysosome fusion. (a) Autophagosomes observed with TEM in cells nontreated (Con) or treated with 100 nM or 200 nM sodium arsenite for 4 h. Arrows indicate autophagosomes. Scale bar is 2 μm (up) and 0.5 μm (down). (b) Number of autophagosomes per cell according to TEM. (c) Western blot for LC3 and p62 in HaCaT cells treated with 100 nM, 200 nM, or 500 nM arsenite for 6 h. (d) Western blot for LC3 and p62 in HaCaT cells treated with 100 nM arsenite at different time points. (e) Protein levels of p62 detected with Western blot. Arsenite-induced inhibition of autophagic flux was tested with chloroquine (CQ, 30 μM) pretreatment and in the absence (-) or presence (+) of 100 nM arsenite for 6 h. (f) Quantification of orange/yellow LC3 puncta in the cell. HaCaT cells were transfected with a tandem mRFP-GFP-LC3 and then treated with 100 nM arsenite for 4 h or 30 μM CQ for 6 h. The number of puncta in cells was counted using ImageJ software. Average number of orange/yellow puncta per cell from 16 randomly selected cells in each group was shown. (g) Representative image of LC3 fluorescence observed by a confocal microscope. Scale bar is 50 μm. (h) Western blot for LAMP1 and LAMP2 in HaCaT cells treated with 100 nM, 200 nM, or 500 nM arsenite for 6 h. (i) Western blot for LAMP1 and LAMP2 in HaCaT cells exposed to 100 nM arsenite at different time points. For Western blot, upper: representative image; lower: quantification of protein levels determined with Western blot. . , compared with Con (or As- and CQ-).

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