Fig. 4: SUMO3 conjugation attenuates ACE2 degradation through autophagy. | Nature Communications

Fig. 4: SUMO3 conjugation attenuates ACE2 degradation through autophagy.

From: Suppression of ACE2 SUMOylation protects against SARS-CoV-2 infection through TOLLIP-mediated selective autophagy

Fig. 4

a 293 T cells were transfected with plasmid encoding Flag-ACE2 together with Myc-PIAS4 vector treated with MG132 (10 μM), 3-methyladenine (10 mM), or bafilomycin A1 (0.2 μM) for 6 h. The cell extracts were analyzed by immunoblotting. b Lysates of Calu-3 cells transfected with scramble or PIAS4-specific siRNA and treated with rapamycin (250 nM) for 24 h, were collected for immunoblot analysis. c hNEPCs were treated with rapamycin (250 nM) (up) or EBSS (down) with indicated time points, and the proteins were harvested for immunoblot analysis. d 293 T cells transfected with scramble siRNA along with RB1CC1- or ATG13-specific siRNAs were transfected with plasmids encoding Flag-ACE2 and Myc-PIAS4, the lysates were analyzed by immunoblotting. e Wild-type (WT), BECN1- and ATG5-knockout (KO) 293 T cells were transfected with vectors of Flag-ACE2 and Myc-PIAS4, the lysates were subjected to immunoblot analysis. f Calu-3 cells were treated with ML-792 (10 μM) (up) or ginkgolic acid (GA) (5 μM) (down) together with 3-MA (10 mM), chlorpromazine (CPZ) (10 μM), methyl-β-cyclodextrin (MβCD) (1 mM), or cytochalasin D (CD) (2 μM), and the proteins were harvested for immunoblot analysis. g Immunoblot analysis of Calu-3 cells transfected with scramble or PIAS4-specific siRNAs and treated with chlorpromazine (CPZ) (10 μM), methyl-β-cyclodextrin (MβCD) (1 mM), or cytochalasin D (CD) (2 μM). h HeLa cells transfected with Flag-ACE2 along with ML-792 (10 μM) or ginkgolic acid (GA) (5 μM) treatment, followed by labeling of ACE2 (green) with specific antibodies. Scale bar, 20 μm. Representative confocal microscopy images from n = 3 independent biological samples for each group. i Total, cytosolic, and membrane fractions of Calu-3 cells pretreated with DMSO or ML-792 (10 μM) were immunoblotted with antibodies directed against ACE2, a cytosol marker (α-tubulin), or a PM marker (E-cadherin). In a–g and i, similar results are obtained by three independent biological experiments. Source data are provided as a Source data file.

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