Fig 2: Ubiquitination of ATG101 is regulated through the C-terminal domain. (a) Cellular ATG101 is degraded primarily via the proteasomal pathway. MIA PaCa-2 cells were plated overnight and incubated with the protein synthesis inhibitor cycloheximide (CHX, 10 μg/mL) alone or with the proteasome inhibitor MG132 (5 μM) or lysosome inhibitor CQ (10 μM) at the time indicated. For western blot analysis, ATG101 levels were normalized to β-actin as the gel loading control. Western blot band intensities were quantified by Image J. Quantification is presented on the graph. Error bars indicate the mean ± SEM of three independent experiments. * p < 0.05. (b) Degradation of ATG101 is associated with ubiquitination. ATG101 KO HEK293T cells were transfected with HA-Ubiquitin (Ub) and/or FLAG-ATG101, and FLAG-ATG101 was immunoprecipitated using anti-FLAG. Immunoprecipitates were analyzed by western blot using anti-HA. (c) Removal of the ATG101 C-terminus (ΔC) reduced the rate of degradation. ATG101 KO MIA PaCa-2 cell were plated overnight and transfected with FLAG vector (Vec), ATG101 full length (FL) or ATG101ΔC (ΔC) for 24–48 h, then incubated in nutrient complete medium or amino acid deprivation medium (–AA) for 4 h prior to western blot analysis. (d) Removal of the ATG101 C-terminus (ΔC) reduced ubiquitination. ATG101 KO HEK293T cells were co-transfected with HA-Ub and FLAG-ATG101 FL or ΔC mutant. FLAG-ATG101 was immunoprecipitated with anti-FLAG, then the immunoprecipitated complexes were analyzed for ATG101 ubiquitination by western blotting using anti-HA. (e) Deletion of the C-terminus stabilized ATG101 protein during CHX treatment. MIA PaCa-2 cells were plated overnight, transfected with FLAG Vector (Vec), ATG101 (FL), or ATG101ΔC (ΔC) for 24 h, and then treated with CHX (10 μg/mL). Cell lysates were collected at the indicated time and immunoblotted for ATG101 and β -actin. ATG101 levels were normalized to β-actin (gel loading control). Quantification is presented on the graph. Error bars indicate the mean ± SEM of three independent experiments. ** p < 0.01. (f) The specific type of ubiquitin tends to conjugate to ATG101. Cells were transfected with ATG101 (FL) and a mutant ubiquitin with only a single lysine (K). The ATG101 promoted conjugation of K48-only ubiquitin. HEK293T cells were transfected with HA-Ub-wild type (WT), K27-only, -K48-only, or -K63-only ubiquitin mutants plus FLAG-ATG101, HA-Ubiquitin was immunoprecipitated using anti-HA, then the immunoprecipitated complexes of HA-Ub were analyzed for ATG101 ubiquitination by western blotting using anti-FLAG or anti-ATG101. Western blot band quantification, ATG101 levels/HA-Ub levels from the immuno-complex subsequent to immunoprecipitation, is presented on the graph. Error bars indicate the mean ± SEM of three independent experiments. * p < 0.05.

Fig 3: Autophagy-related protein 101 (ATG101) is required for autophagy to promote cell growth and survival. (a) Knockout of ATG101 (sgATG101) in MIA PaCa-2 cells using the Lenti-CRISPR system reduced proliferation rate compared to Control (sgCTL) MIA PaCa-2 cells as measured by the IncuCyteTM analyzer. MIA PaCa-2 cells stably expressing GFP-LC3 were used. Cell confluence levels were measured in real-time and expressed as a proportion (%) of complete cell coverage. Error bars indicate the mean ± SEM of three independent experiments. (b) Knockout of ATG101 in MIA PaCa-2 cells also enhanced apoptotic death rate under nutrient deprivation as measured by Annexin V/propidium iodide (PI) staining and flow cytometry. WT or ATG101 KO MIA PaCa-2 cells were plated overnight and then incubated in HBSS or the medium without amino acids (amino acid deprivation, −AA) for 48 h. Error bars indicate the mean ± SEM of three independent experiments. * p < 0.05; *** p < 0.001. (c) ATG101 knockout (KO) altered the levels of autophagy markers as measured by western blotting. Cell lysates prepared from Control (sgCTL) or ATG101 KO (sgATG101) stably expressing GFP-LC3 MIA PaCa-2 cells cultured in complete medium(COM) and in amino acid-deprived medium (–AA) for 4 h, were immunoblotted for ATG101, p62, LC3B, and β-actin (gel loading control). (d) ATG101 KO reduced the formation of autophagosomes as measured by GFP-LC3 puncta. Control (sgCTL) or ATG101 KO (sgATG101) MIA PaCa-2 cells stably expressing GFP-LC3 were seeded overnight and subsequently starved in amino acid-deprived medium (–AA) for 4 h. Cell nuclei were stained with Hoechst 33342, and images were acquired using a confocal fluorescence microscope. At least five distinct regions were imaged per condition and quantified. Scale bar: 20 µm. GFP-LC3 puncta area was normalized to the Hoechst 33,342 stained area and is presented as a percentage on the quantification graph. Error bars indicate the mean ± SEM of three independent experiments. * p < 0.05; *** p < 0.001.

Fig 4: ATG101-mediated autophagy supported while HUWE1-catalyzed ubiquitination of ATG101 reduced cancer cell survival. (a) Knockdown of HUWE1 reduced the apoptotic death of cancer cells under nutrient starvation as assessed by Annexin V/propidium iodide (PI) staining and flow cytometry. shCTL or shHUWE1 MIA PaCa-2 cells were plated overnight and incubated in amino acid deprivation (–AA) medium for 48 h. Error bars indicate the mean ± SEM of three independent experiments. *** p < 0.001. (b) Additional knockdown of ATG101 and/or WIPI2 further enhanced cell death in HUWE1 knockdown cells. shCTL or shHUWE1 MIA PaCa-2 cells were transfected with WIPI2 and/or ATG101 siRNA for 24 h and subsequently incubated in HBSS for 24 h. Cell death was analyzed as in (a). Error bars indicate the mean ± SEM of three independent experiments. * p < 0.05; *** p < 0.001. (c) Schematic diagram of HUWE1 targeting ATG101 for ubiquitination and degradation, promoting cancer cell death via blocking autophagy activation under metabolic stress.

Fig 5: HUWE1 mediates ATG101 ubiquitination and degradation. (a) HUWE1 knockdown (KD) in MIA PaCa-2 cells increased ATG101 protein levels as measured by western blotting. MIA PaCa-2 cells were incubated with HUWE1 siRNA for 24 h and then incubated with CHX (10 μg/mL) at the time indicated. Cell lysates were analyzed by western blotting using the indicated antibodies. Quantification and statistical analysis were as in Figure 2a. (b) HUWE1 KD reduced ATG101 ubiquitination. HEK293T cells were transfected with siRNAs targeting ATG101 E3 ligase candidates (siCTL, siCHIP, siHUWE1, siTRAF6) for 24 h, then co-transfected with HA-Ub and FLAG-ATG101 for 24 h. FLAG-ATG101 was immunoprecipitated with anti-FLAG. Immunoprecipitates were analyzed by western blot using anti-HA for determining ATG101. * non-specific bands. (c) HUWE1 KD reduced ATG101 ubiquitination. shCTL or shHUWE1 HEK293T cells were co-transfected with HA-Ub and FLAG-ATG101. HA-ubiquitin was immunoprecipitated with anti-HA, then the immunoprecipitated complexes were analyzed for ATG101 ubiquitination by western blotting using anti-FLAG. * non-specific bands. (d,e) Co-immunoprecipitation demonstrates a possible physical interaction between HUWE1 and ATG101 in MIA PaCa-2 cells. Endogenous HUWE1 was immunoprecipitated using anti-HUWE1 and the immunoprecipitates were analyzed by western blot using anti-ATG101. Cells were cultured in (d) basal condition and (e) starvation condition for incubating cells with amino-acid medium –AA) for 2 h. * non-specific bands. (f) A physical interaction between HUWE1 and ectopic expressed ATG101 in both basal and nutrient deprivation. MIA PaCa-2 cells were transfected with FLAG-ATG101 for 48 h. Cellular starvation, immunoprecipitation, and subsequent western blot analysis were as described in Figure 3d,e. * non-specific bands.