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

Figure 6

HaCaT cells were treated with or without 50 mJ/cm² UVB and then incubated in the presence or absence of 20 nM Rapamycin, 100 nM everolimus, 1 μM Torin 1, or 1 μM pp242 for 12 hours. Cytotoxicity measurement was performed using Cell Counting Kit-8 (a). Western blotting analysis was performed using primary antibodies against PARP, cleaved PARP, caspase-3, and cleaved caspase-3 (b). GAPDH served as a loading control. The cells were imaged for Annexin V-EGFP apoptosis detection using a laser scanning confocal microscope (c). The percentages of cells marked by Annexin V-EGFP with or without PI (d) or single PI (e) were calculated. The individual experiment was performed three times, and the results were obtained for statistical analysis. Representative figures were shown from three independent experiments. Bars = 20 μm. Rapa: Rapamycin; Ever: everolimus. NS: nonsense. .

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