Fig. 3
UVB and ZnONPs caused severe cell damage and subsequent lysosome dysfunction to induce autophagic flux blockage. A HaCaT cells were examined after 24 h using a trypan blue dye exclusion assay. Histograms show the percentages of in the control, UVB irradiation alone (40, 50, 68 mJ/cm2), ZnONPs alone (5, 10, 12.5, 15 μg/ml) and combined treatment groups. (*p < 0.05, **p < 0.01, ***p < 0.001 compared with control, #p < 0.05, ##p < 0.01, ###p < 0.001 compared with UVB alone) B ZnONPs, UVB and combination treatment induced autophagy in HaCaT cells at 24 h. Flow cytometry was used to detect green (FL-1) and red (FL-3) fluorescence in AO-stained cells. C Histograms show the percentages of autophagic cells after the different treatments. The data are presented as the mean ± SD from three independent experiments (*p < 0.05, **p < 0.01, compared with control, #p < 0.05, ###p < 0.001 compared with UVB alone). D Fluorescence microscope was used to visualize the acidic vesicular organelles (AVOs; red fluorescence) as well as the cytoplasm and nucleus (green fluorescence) after the AO staining. E Histogram represents the quantification of the AVOs red fluorescence (***p < 0.001, compared with control, ###p < 0.001 compared with UVB alone). F Measurement of autophagy-related proteins in HaCaT cells after UVB and ZnONP treatment for 24 h. G Immunofluorescence assays with LC3 (green) and LAMP-1 (red) antibodies were performed to identify the sites of autophagosomes and lysosomes. Scale bars: 50 μm. The fusion of autophagosomes with lysosomes was blocked when cells were exposed to ZnONPs for 24 h. H Fluorescence assays of R6G-labeled ZnONPs and lysosomal activity in the cells under a time course of combined treatment. Lysosomal activity was detected by LysoSensor DND-189 staining. Scale bars: 25 μm. DAPI staining showed the nuclear DNA of cells. I Cytosolic release of lysosomal proteases was detected by western blotting. HaCaT cells were treated with UVB alone and UVB + ZnONPs for the indicated times, followed by cell fractionation to obtain cytosolic and membrane fractions. LAMP-1 is a lysosomal membrane marker, and GAPDH is a cytosolic marker. There was no cross-contamination of cytosolic and lysosomal fractions when we separated the cell fractions. Cathepsin B was detected in the cytosolic fraction after 6 h of exposure to UVB + ZnONPs and was consistently expressed after 24 h of exposure