Impact on Autophagy and Ultraviolet B Induced Responses of Treatment with the MTOR Inhibitors Rapamycin, Everolimus, Torin 1, and pp242 in Human Keratinocytes

Figure 3

HaCaT cells were treated with or without 1 μM Torin 1 (a) or 1 μM pp242 (d) for 12 hours in the presence or absence of E64d and pepstatin. Then, the cell lysate was subjected to determine the level of LC3 protein by western blotting. GAPDH served as a loading control. The ratios of LC3-II/GAPDH were calculated, and statistical differences between treatment and nontreatment (NT) were analyzed. HaCaT cells were pretreated with or without GFP-LC3B before Torin 1 (b) or pp242 (e) treatment for 12 hours in the presence or absence of E64d and pepstatin. HaCaT cells were treated with or without Torin 1 (c) or pp242 (f) for 12 hours in the presence or absence of E64d and pepstatin. Then, cells were incubated with AO. The cells (b, e, c, and f) were imaged by a laser scanning confocal microscope, and the means of GFP-LC3 puncta or red/green fluorescence ratios for individual cells were determined for statistical analysis. The data were shown as means ± SD from three independent experiments and the representative figures were shown. Bars = 20 μm. NS: nonsense.

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