Journal: Nature Communications

Article Title: Oxygen-sensitive methylation of ULK1 is required for hypoxia-induced autophagy

doi: 10.1038/s41467-022-28831-6

Figure Lengend Snippet: b – e , h – k Immunoblot were performed with the indicated antibodies. a Indicated cells were incubated under 1% oxygen for 12 h. Flag-ULK1 proteins were precipitated, and ULK1 kinase activity was analyzed. Data represent the mean ± SD from three independent experiments. P -values are from the two-sided t -tests. Bonferroni correction was used for multiple hypothesis correction. b Indicated LN229 cells incubated under 1% oxygen for 12 h. Exo, exogenous; Endo, endogenous. c Purified ULK1 proteins was immobilized on beads, and mixed with purified His-PRMT5 protein for an in vitro methylation assay. Immobilized ULK1 proteins were washed, and mixed with purified GST-Atg13 or His-Beclin 1 proteins for an in vitro kinase assay. d LN229 cells with expression of Flag-ULK1 were incubated under 1% oxygen for 12 h. Flag-ULK1 proteins were pulldown, washed and mixed with purified WT His-KDM5C or His-KDM5C H514A protein for an in vitro demethylation assay under normoxia condition. The ULK1 precipitates were washed, and mixed with purified GST-Atg13 or His-Beclin 1 proteins for an in vitro kinase assay. e Indicated cells were incubated under 1% oxygen for 12 h. VPS34 activity in the precipitates were measured. Data represent the mean ± SD from three independent experiments. P -value is from the two-sided t -test. f Indicated cells were incubated under 1% oxygen for 12 h. EGFP-FYVE puncta formation was analyzed. Scale bar, 6 µm. Data represent the mean ± SD from three independent experiments. P -value is from the two-sided t -test. g Human ULK1 structure (PDB code: 4WNP) shows an activation loop domain (yellow). The surrounding areas of R170 and T180 are boxed and enlarged. For the side chain of R170 and T180, nitrogen, oxygen and carbon atoms were shown in purple, red and green, respectively. h LN229 cells were incubated under 1% oxygen. i Indicated cells were incubated under 1% oxygen for 12 h. j , k Purified WT Flag-ULK1 ( j ) or/and Flag-ULK1 R170K ( k ) proteins were subjected to a His-PRMT5 protein-mediated in vitro methylation assay and an in vitro autophosphorylation assay. Source data are provided as a Source data file.

Article Snippet: Antibodies recognizing ULK1 (#8054, 1:1000), HIF1α (#36169, 1:1000), Tubulin (#2125, 1:2000), LC3B (#83506, 1:1000), p62 (#88588, 1:1000), Beclin 1 (#4122, 1:1000), Beclin 1 pS15 (#84966, 1:500), Atg13 (#13468, 1:1000), Atg13 pS355 (#46329, 1:500), S6K pT421/pS424 (#9204, 1:1000), Tomm20 (#42406, 1:500), TSC1 (#6935: 1:500) and AMPKα (#5832, 1:1000) were obtained from Cell Signaling Technology.

Techniques: Western Blot, Incubation, Activity Assay, Purification, In Vitro, Methylation, Kinase Assay, Expressing, Demethylation Assay, Activation Assay

Journal: Biology

Article Title: Leelamine Modulates STAT5 Pathway Causing Both Autophagy and Apoptosis in Chronic Myelogenous Leukemia Cells

doi: 10.3390/biology11030366

Figure Lengend Snippet: Effect of LEE in combination with pharmacological blockers. ( A ) KBM5 cells (5 × 10 5 cells/well) were treated with autophagy inhibitor, 1 mm of 3-Methyladenine (3-MA) and 2 μm of LEE for 48 h. Cells were stained with acridine orange and analyzed by cell flow cytometry. ( B ) 3-MA and LEE treated KBM5 cells were evaluated for expression of various autophagy related proteins. ( C ) KBM5 cells (5 × 10 5 cells/well) were treated with caspase-3 inhibitor, 50 μm of Z-DEVE-FMK and 2 μm of LEE for 48 h. The cells were labelled with annexin-FITC for 15 min, and then analyzed by cell flow cytometry. ( D ) Z-DEVE-FMK and LEE treated KBM5 cells were examined for caspase-3 and PARP expression by Western blot. ( E ) Beclin-1 and Atg7 proteins were knocked down by siRNA transfection. ( F ) The expression of Beclin-1 or Atg7 proteins in KBM5 cells was knocked out by siRNA transfection. The cells were then treated with LEE (2 μm) for 48 h and LC3 levels were analyzed by Western blot. ( G ) The cells were processed as described in F and then subjected to AO assay.

Article Snippet: Western blot membrane of LC3, Atg7, p-beclin-1, beclin-1, and b-actin proteins were detected with anti-LC3 (12741s; 1:3000; Cell signaling), anti-Atg7 (85585s; 1:3000; Cell signaling), anti-p-beclin-1 (84966s; 1:3000; Cell signaling), anti-beclin-1 (4122s; 1:3000; Cell signaling), and anti-b-actin (sc47778; 1:5000; Santacruz) antibodies.

Techniques: Staining, Flow Cytometry, Expressing, Western Blot, Transfection

Journal: Journal of Virology

Article Title: Inhibition of Autophagy Suppresses SARS-CoV-2 Replication and Ameliorates Pneumonia in hACE2 Transgenic Mice and Xenografted Human Lung Tissues

doi: 10.1128/JVI.01537-21

Figure Lengend Snippet: Autophagy induced by SARS-CoV-2 promotes virus replication in Vero E6 cells via the VPS34 complex. SARS-CoV-2-infected (MOI, 0.008) Vero E6 cells were harvested at different time points (0 h, 3 h, 6 h, 12 h, 24 h, 36 h, 48 h, and 72 h) after infection. (A to F) Alterations in the signaling pathway involved in the cellular autophagy machinery were analyzed by Western blotting, and this included the Akt-mTOR pathway (A), AMPK-TSC2/Raptor pathway (B), p-ULK1 (Ser 757), p-Atg13 (Ser 355), Atg13 proteins (C), VPS34-VPS15-Beclin1 complex (D), Atg14 protein (E), and phagophore and autophagosome membrane-associated proteins (F). (G) Viral load in SARS-CoV-2-infected Vero E6 cells pretreated with SAR405 (1 μM) or 3-MA (5 mM). Cell samples were collected at 0 h, 24 h, 48 h, and 72 h after infection and analyzed by RT-qPCR. (H) Viability of Vero E6 cells treated with 3-MA after SARS-CoV-2 infection (MOI, 0.008) for 72 h. (I) Western blotting of Atg14 knockdown efficiency in Vero E6 cells. (J) Viral load in SARS-CoV-2-infected Vero E6 cells transfected with control or Atg14 siRNA. Samples were harvested at 24 hpi. (K) Western blotting of alterations in Atg5-related proteins in Atg5 −/− Vero E6 cells. (L) Viral load in SARS-CoV-2-infected Atg5 +/+ and Atg5 −/− Vero E6 cells at 24 hpi. Data were expressed as means ± SEM from three independent experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Article Snippet: Antibodies against Atg5 (2630), p-AKT (4060), AKT (4685), p-mTOR (5536), mTOR (2983), p-S6 (4858), S6 (2317), p-4E-BP1 (2855), 4E-BP1 (9644), p-AMPK (2535), AMPK (5831), p-TSC-2 (3614), TSC-2 (4308), p-Raptor (2083), Raptor (2280), p-Atg13 (26839), Atg13 (13273), ULK1 (8054), p-ULK1 (Ser757) (14202), p-ULK1 (Ser555) (5869), VPS15 (14580), VPS34 (4263), Beclin1 (3495), p-Beclin1 (84966), Atg14 (96752), Atg16L1 (8089), p62 (16177), and GAPDH (5174) were obtained from Cell Signaling Technology (Danvers, MA).

Techniques: Infection, Western Blot, Quantitative RT-PCR, Transfection