Journal: Autophagy

Article Title: RNF186 regulates EFNB1 (ephrin B1)-EPHB2-induced autophagy in the colonic epithelial cells for the maintenance of intestinal homeostasis

doi: 10.1080/15548627.2020.1851496

Figure Lengend Snippet: EFNB1-induced autophagy is independent of MTOR inhibition. (A) Ls174t cells were treated as indicated, followed by western blot analysis of the indicated proteins. Densitometry quantification from three independent experiments was shown in the right panel. (B-D) Ls174t cells were pretreated with SBI-0206965 (10 μM) (B), 3-MA (5 mM) (C) or wortmannin (100 nM) (D) for 1 h, followed by the treatment of plate-coated Fc or ephrin-B1-Fc (10 ug/ml) in presence of the inhibitors for the indicated time. Cell lysates were analyzed by western blot for the indicated proteins. Densitometry quantification from three independent experiments was shown in the right panel. (E) Ls174t cells were pretreated with DMSO, 3-MA (5 mM) or wortmannin (100 nM) for 1 h, followed by plate-coated Fc or ephrin-B1-Fc (10 μg/ml) for 2 h in presence of the inhibitors and followed by confocal microscopy analysis of MAP1LC3B puncta. Quantification of cells with puncta of MAP1LC3B-II was shown in the bottom panel. Scale bar: 5 μm. (F) Ls174t cells were pretreated with VPS34-IN1 (2 μM) for 1 h, followed by the treatment of plate-coated Fc or ephrin-B1-Fc (10 ug/ml) in presence of the inhibitor for the indicated time. Cell lysates were analyzed by western blot for the indicated proteins. Densitometry quantification was shown in the right panel. (G) Control-gRNA or ATG5-gRNA infected Ls174t cells were treated with 10 μg/ml plate-coated Fc (0 time point) or ephrin-B1-Fc, followed by western blot analysis of the indicated proteins. Densitometry quantification from three independent experiments was shown in the right panel. WT: wild type; KO: knockout; 3-MA: 3-methyladenine. All error bars represent SEM of technical replicates. *: P < 0.05, **: P < 0.01, ***: P < 0.001, ****: P < 0.0001 based on two-sided unpaired T test (A-G). Densitometry quantification of A-D and F-G were based on three independent experiments. Data are representative of three independent experiments

Article Snippet: ULK inhibitor SBI-0206965 (T2128), PtdIns3K inhibitor 3-MA (T1879), wortmannin (T6283) and Vps34-IN-1 (T7015) were bought from TargetMol.

Techniques: Inhibition, Western Blot, Confocal Microscopy, Infection, Knock-Out

Journal: Virologica Sinica

Article Title: Cytoplasmic domain and enzymatic activity of ACE2 are not required for PI4KB dependent endocytosis entry of SARS-CoV-2 into host cells

doi: 10.1016/j.virs.2022.03.003

Figure Lengend Snippet: S pseudovirus transduces Huh 7 cells via clathrin-mediated endocytosis. A Huh 7 cells pretreated with different concentrations of camostat mesylate (5, 15, 50 μmol/L) or E-64d (5, 10, 25 μmol/L) were transduced with S pseudovirus and luciferase was measured. Values were normalized to control and expressed as mean ± SD. B Huh 7 cells pretreated with lysosomotropic agents (50 mmol/L NH 4 Cl, 100 μmol/L chloroquine and 100 nmol/L bafilomycin A) were transduced with S pseudovirus or VSV-G pseudovirus. Luciferase was measured to indicate viral infection. Values were normalized to control and expressed as mean ± SD. C Immunostaining of S pseudovirus infected Huh 7 cells for Spike (red) and EEA1 (green). Scalebar, 10 μm. D Huh 7 cells pretreated with indicated concentrations of pitstop were transduced with S pseudovirus or VSV-G pseudovirus and luciferase was measured. Values were normalized to control and expressed as mean ± SD. E Huh 7 cells pretreated with indicated concentrations of CPZ were transduced with S pseudovirus or VSV-G pseudovirus and luciferase was measured. Values were normalized to control and expressed as mean ± SD. F Huh 7 cells stably expressing a negative control or three independent CHC sgRNAs with Cas9 were transduced with S pseudovirus and luciferase was measured. Values were normalized to control and expressed as mean ± SD. Inset, immunoblotting of CHC expression in Huh 7 cells as described above. S, spike; CQ, chloroquine; Baf A, bafilomycin A; CPZ, chlorpromazine; CHC, clathrin heavy chain; SD, standard deviation.

Article Snippet: PIK93 (#HY-12046), Wortmannin (#HY-10197), camostat mesylate (#HY-13512) and E-64d (#HY-100229) were purchased from MedChemExpress.

Techniques: Transduction, Luciferase, Control, Infection, Immunostaining, Stable Transfection, Expressing, Negative Control, Western Blot, Standard Deviation