Fig 2: Severe Acute Respiratory Syndrome Coronavirus-2 ORF3a targets UVRAG to modulate PI3KC3 complex formation. (A) SARS-CoV-2 ORF3a interacts with UVRAG in yeast two-hybrid system. (B) SARS-CoV-2 ORF3a binds to UVRAG in HEK293T cells. HEK293T cells were co-transfected with indicated plasmids and cell lysates were collected and subjected to immunoprecipitation (IP) and IB with indicated antibodies at 48 h post-transfection. (C) Schematic diagram of SARS-CoV-2 ORF3a-UVRAG interaction. (D) The C2 and CCD of UVRAG bind to SARS-CoV-2 ORF3a. (E) SARS-CoV-2 ORF3a co-localizes with endogenous UVRAG. HeLa-ORF3a stable cells were subjected to immunostaining with antibodies against Flag or UVRAG. Scale bar, 15 μm. (F) SARS-CoV-2 ORF3a binds to endogenous UVRAG in HEK293T cells. (G–I) SARS-CoV-2 ORF3a blocks PI3KC3-C2 but facilitates PI3KC3-C1. HEK293T cells were co-transfected with indicated plasmids and cell lysates were collected and subjected to IP and IB with indicated antibodies at 48h post-transfection.

Fig 3: As ceRNA targets of SLC7A11, (A) STX17, (B) UVRAG, and (C) RAB33B were positively co-expressed in 12, 20 and 12 kinds of cancers according to pan-cancer analyses. The other 2 cancers that SLC7A11 negatively co-expressed with RAB33B was excluded. The gene expression values from RNA-seq data were presented as log2 (FPKM + 0.01) (P<0.05). BRCA, Breast Invasive Carcinoma; THCA, Thyroid Carcinoma; COAD, Colon Adenocarcinoma; PRAD, Prostate Adenocarcinoma; UCEC, Uterine Corpus Endometrial Carcinoma; READ, Rectum Adenocarcinoma; HNSC, Head and Neck Squamous Cell Carcinoma; LGG, Brain Lower Grade Glioma; ACC, Adrenocortical Carcinoma; UVM, Uveal Melanoma; KICH, Kidney Chromophobe; KIRC, Kidney Renal Clear Cell Carcinoma; SKCM, Skin Cutaneous Melanoma; LUSC, Lung Squamous Cell Carcinoma; TGCT, Testicular Germ Cell Tumors; BLCA, Bladder Urothelial Carcinoma; LUAD, Lung Adenocarcinoma; LAML, Acute Myeloid Leukemia; CESC, Cervical Squamous Cell Carcinoma and Endocervical Adenocarcinoma; KIRP, Kidney Renal Papillary Cell Carcinoma; UCS, Uterine Carcinosarcoma; THYM, Thymoma; LIHC, Liver Hepatocellular Carcinoma.

Fig 4: Intermittent fasting restores autophagy in DOX-induced cardiotoxicity.(a) Representative images of LC3 immunohistochemistry in LVs on heart sections from fed or fasted WT and UVRAG-deficient mice 5 days after acute DOX or vehicle treatment. Scale bar: 40 μm. (b) Quantification of LC3-positive dots in LVs in the experiments as illustrated in (a). n = 3 mice for each group. (c) Representative images of LC3 immunohistochemistry in LVs on heart sections from fasted WT and UVRAG-deficient mice at 4 weeks of DOX treatment in chronic cardiotoxicity. Scale bar: 40 μm. (d) Quantification of LC3-positive dots in LVs in the experiments as illustrated in (c). n = 3 mice for each group. (e) Western blot detection of LC3 and p62 in LVs from fed or fasted WT and UVRAG-deficient mice 5 days after acute DOX or vehicle treatment. (f) Quantification of LC3 II protein abundance in the experiments as illustrated in (e). n = 3 mice for each group. (g) Quantification of p62 protein abundance in the experiments as illustrated in (e). n = 3 mice for each group. (h) Western blot analysis of LC3 II and p62 in LVs from fed or fasted WT and UVRAG-deficient mice at 4 weeks of DOX treatment in chronic cardiotoxicity. (i) Quantification of LC3 II protein abundance in the experiments as illustrated in (h). n = 3 mice for each group. (j) Quantification of p62 protein abundance in the experiments as illustrated in (h). n = 3 mice for each gorup. *P < 0.05 vs. WT + DOX + Fed, #P < 0.05 vs. UVRAG−/− + DOX + Fed, §P < 0.05 vs. WT + DOX + Fasted.

Fig 5: UVRAG-associated class III lipid kinase is activated by insulin.(a) ATG14- and UVRAG-containing Vps34 complexes were immunoprecipitated from Hepa1.6 cells grown in the presence (NR) or absence of amino acids (−AA) and assayed for lipid kinase activity. Inputs for each assay were immunoblotted to determine the amounts of the Vps34 co-immunoprecipitated. PI3P signals were densitometrically measured and normalized to Vps34 protein levels. The data are presented as a fold difference of PI3P normalized to co-immunoprecipitated Vps34 levels revealed by immunoblot for each condition. Data are means±s.e.m. (n=4, *P<0.05 versus NR, two-tailed, unpaired Student's t-test). (b) ATG14- and UVRAG-containing Vps34 complexes were immunoprecipitated from primary hepatocytes, which were serum starved for 24 h followed by stimulation with 1 μM insulin (Ins) and assayed for lipid kinase activity. PI3P signals were densitometrically measured. The data are presented as a fold difference of PI3P normalized to co-immunoprecipitated Vps34 levels revealed by immunoblot for each condition. Data are means±s.e.m. (n=3, *P<0.05 versus starved cells, two-tailed, unpaired Student's t-test). Representative immunoblot analyses of ATG14, UVRAG and Vps34 co-immunoprecipitates are presented as Supplementary Fig. 3a and Supplementary Fig. 3b. (c) Primary hepatocytes were transiently transfected with UVRAG–Flag cDNA-expressing vector 12 h post-plating and were kept in the serum containing media. The 36-h post-transfection cells were collected and UVRAG–Flag complexes were immunoprecipitated with anti-Flag antibody. The presence of IRβ and class III PI3K subunits in the immunoprecipitation eluates was revealed by immunoblot. (d) Endogenous IRβ was immunoprecipitated from primary hepatocytes, which were serum starved for 24 h and stimulated with 1 μM insulin. The presence of Vps15 and IRβ in the immunoprecipitation eluates was revealed by immunoblot. Densitometric analyses of co-immunoprecipitated endogenous Vps15 normalized to IRβ are presented as fold difference over the unstimulated condition. Data are means±s.e.m. (n=3, *P<0.05 versus starved cells, two-tailed, unpaired Student's t-test). (e) UVRAG-containing complexes were immunoprecipitated from primary hepatocytes, which were serum starved for 24 h followed by stimulation with 1 μM insulin for indicated times. Immunoprecipitated endogenous class III PI3K subunits were revealed by immunoblotting. Densitometric analyses of co-immunoprecipitated endogenous Rubicon normalized to UVRAG are presented as fold difference over the unstimulated condition. Data are means±s.e.m. (n=3, *P<0.05 versus starved cells, two-tailed, unpaired Student's t-test). The protein G beads served as a control of the nonspecific binding in c–e.