Fig. 3

SR-T100 induced a greater autophagic response in primary epithelial cells expressing HPV16-E6/E7 and HPV18-E6/E7. a Immunoblotting for LC3-I and LC3-II using lysates from primary epithelial cells expressing different types of HPV-E6/E7 treated with or without SR-T100 (5 μg/ml) for 12 h. Similar results were observed in at least three independent experiments. The number indicates the fold change in the LC3-II/actin ratios in cells treated with SR-T100 versus those without SR-T100 treatment. b Activation of autophagy was observed in primary epithelial cells expressing different types of HPV-E6/E7 that were treated with SR-T100 (5 μg/ml) for 12 h using GFP-LC3 accumulation. The cells were first transfected with LC3-GFP, infected with different types of Lenti-HPV-E6/E7 viruses, and then treated with SR-T100 for 12 h. GFP-LC3 accumulation was analyzed under a fluorescence microscope (400×), followed by quantitation of the number of green spots per cell showing LC3 accumulation in cytoplasmic vacuoles (inset panel). (***p < 0.001: HPV16 or 18 versus HPV6b or 11). Representative images of cells that were treated with SR-T100 for 12 h are shown. c Visualization of the activation of autophagy in primary epithelial cells expressing different types of HPV-E6/E7 that were treated with SR-T100 (5 μg/ml) for 12 h using MDC staining. The formation of autophagic vacuoles was suggested by the interspersed MDC labeling in the cytoplasm observed under a fluorescence microscope (400×). Representative images of cells that were treated with SR-T100 for 12 h are shown. Quantitative results of MDC staining from three independent experiments (**p < 0.001: HPV16 or HPV18 versus HPV6b or HPV11). d Ultrastructural images of the SR-T100-treated cells were obtained using an electron microscope. Primary epithelial cells were infected with or without HPV6b-E6/E7 or HPV16-E6/E7 lentiviruses for 24 h and treated with SR-T100 (5 μg/ml) for 12 h. The right panel (25,000×) shows the magnified image of the area indicated by the box in the left panel (8,000×). Autophagic vacuoles are denoted by arrows. Note the increased number of autophagic vacuoles in cells expressing HPV16-E6/E7. N, nucleus