Fig. 5: SCFD1 regulates autophagosome-lysosome fusion.

A The number of LC3 puncta clearly increased upon SCFD1 knockdown. U2OS cells were transfected with indicated the siRNAs. Cells were cultured in full medium stimulated with Torin. LC3 puncta were detected using an LC3 antibody and observed via confocal microscopy. Scale bar, 10 μm. B Quantification of LC3 puncta per cell. One-way ANOVA (Data are shown as the mean ± SEM. **p < 0.01., ****p < 0.0001, n = 30). C SCFD1 knockdown blocks autophagy flux. U2OS cells transfected with either siSCFD1 or control siRNAs were incubated with full medium, full medium with Torin, or full medium with Torin and chloroquine (CQ). The LC3 was detected by western blotting. D The extent of LC3/LAMP2 overlap was reduced upon siRNA-mediated knockdown of SCFD1. Immunofluorescence of endogenous LC3 and Lamp2 was observed in U2OS cells with the indicated siRNAs, with or without Torin treatment. Scale bar, 10 μm. E SCFD1 depletion results in autophagosome accumulation. mRFP-GFP-LC3 assays performed in U2OS cells transfected with the indicated siRNAs, with or without Torin1 stimulation. Scale bar, 10 µm. F Quantification of data from part G. The ratio of GFP negative mRFP positive LC3 puncta versus both GFP and mRFP positive LC3 puncta. One-way ANOVA (Data are shown as the mean ± SEM. ****p < 0.0001, n = 58). G Localization of VAMP8 and STX17 is dependent on SCFD1. Immunofluorescence of U2OS cells expressing GFP-VAMP8 and Flag-STX17, transfected with indicated siRNAs, treated with or without Torin1. Scale bar, 5 µm. No overlap between STX17 and VAMP8 was detected upon siRNA-mediated knockdown of SCFD1.