Decreased IL-1β levels observed in HPV16 E6-positive cells are independent of autophagy or lysosomal degradation.
A) Confocal microscopy analysis after immunostaining of IL-1β (red) in immortalized keratinocytes after transduction with a GFP-tagged LC3-expressing lentivirus (LC3-GFP, green). GFP-LC3-positive cells were treated for 6 h with 1 mM 3-Methyladenine (3-MA) to block autophagy. The scale bars represent 10 µm. B) ELISA of intracellular IL-1β derived from immortalized HPV-positive cells treated for 6 h with 100 nM of bafilomycin to inhibit autophagosome maturation or with 1 mM of 3-MA to block autophagy. C) Quantification of the lysosomal activity (measured in mean fluorescence intensity, MFI) by confocal microscopy in immortalized HPV-positive cells. Cells were incubated with DQ™ Red-BSA for 8 h in the presence or absence of 100 nM bafilomycin. Nuclei (blue) were stained using Hoechst dye solution. The scale bar represents 10 µm. D) Quantification of cathepsin B activity (expressed as mean fluorescence intensity, MFI) by confocal microscopy in untreated HPV-positive cells stained with the specific fluorogenic cathepsin B substrate Magic Red™. Nuclei (blue) were stained using Hoechst dye solution. Scale bars represent 10 µm. E) ELISA of intracellular IL-1β from immortalized HPV-positive cells treated for 6 h with 25 µM cathepsin B inhibitor (CA-074 Me), 10 µM of lysosomal protease inhibitor (leupeptin), 20 µM of calpain inhibitor (PD150.606) or 20 µM of lysosome fusion inhibitor (vincristine). The analyses in C and D were performed using high throughput high resolution fluorescent microscopy analysis (BD pathway) in combination with a cell imaging analysis program (CellProfiler). The graphs show mean levels of five independent experiments each performed with 10.000 events/well per experiment (± SEM) ANOVA ***p<0.001. The bar graphs shown in B and E represent the standard error of the mean (± SEM) ANOVA ***p<0.001, NS: non-significant statistical differences.