Fig 1: HUWE1 suppresses autophagy by regulating ATG101 and WIPI2 levels (a) HUWE1 KD enhanced GFP-LC3 puncta formation in MIA PaCa-2 cells. shCTL or shHUWE1 MIA PaCa-2 cells stably expressing GFP-LC3 were plated overnight, and subsequently incubated with rapamycin (0.5 μM) for 4 h prior to imaging. Cell imaging, quantification and statistical analysis were as described in Figure 1d. * p < 0.05. (b) HUWE1 KD showed enhancement of autophagy activity as indicated by western blotting for autophagy marker proteins. Cell lysates from (a) were analyzed by western blot using the indicated antibodies for autophagy marker proteins. Quantification and statistical analysis were as in Figure 1c. (c) ATG101 KD reversed the increase in GFP-LC3 puncta formation induced by shHUWE1 cells. shCTL or shHUWE1 MIA PaCa-2 cells stably expressing GFP-LC3 were transfected with ATG101 siRNA for 48 h then incubated with rapamycin (0.5 μM) for 4 h. Cell imaging, quantification and statistical analysis were as in Figure 1d. Scale bar: 20 μm. Error bars indicate the mean ± SEM of three independent experiments. ** p < 0.01; *** p < 0.001. (d) Both ATG101 and WIPI2 double KD reversed the increase in autophagy flux induced by shHUWE1. shHUWE1 HeLa cells stably expressing mCherry-GFP-LC3 were transfected WIPI2 and/or ATG101 siRNA for 48 h, followed by incubation in amino acid-deprived medium (–AA) for 4 h. Cells were imaged under starvation conditions as in (c). Scale bar: 20 μm. mCherry puncta area (%) was normalized to Hoechst-stained area and is presented as a percentage on the quantification graph. Error bars indicate the mean ± SEM of three independent experiments. ** p < 0.01 (e) A physical interaction between WIPI2 and ATG101. HEK293T cells were co-transfected with GFP-WIPI2 and FLAG-ATG101 and incubated for 24–48 h in the treatment with vehicle or rapamycin (0.5 μM) for 4 h prior to harvesting. GFP-WIPI2 was immunoprecipitated with anti-GFP, then the immunoprecipitated complexes were analyzed for interaction between ATG101 and WIPI2 by western blotting using anti-FLAG. (f) Removal of the ATG101 C-terminus domain (ΔC) reduced the interaction between ATG101 and WIPI2. HEK293T cells were co-transfected with GFP-WIPI2 and FLAG-ATG101 FL or ΔC for 24–48 h and incubated for 4 h in complete medium (COM) or amino-acid deprived medium (–AA) for starvation. FLAG-ATG101 was immunoprecipitated with anti-FLAG, then immunoprecipitated, analyzed by western blotting using anti-WIPI2.