Fig. 3: SUMOylation promotes the stabilization of ACE2.

a, b Protein abundance (up) and mRNA level (down) of ACE2 in Calu-3 cells treated with indicated concentration of ML-792 (a) or 2-D08 (b) for 24 h. c 293 T cells expressing wild-type (WT) Flag-ACE2 or its indicated mutants were treated with 2-D08 (200 μM) for 24 h. Different amounts of vectors for WT and mutants of Flag-ACE2 were transfected into 293 T cells for equal expression. The lysates were analyzed with immunoblotting. d Overview of the screening for functional deSUMOylating enzymes in human ACE2-expressing A549 cells with the infection of SARS-CoV-2 S pseudotyped virus. e Luciferase activity in control and deSUMOylating enzyme depletion hACE2-expressing A549 cells challenged with SARS-CoV-2 S pseudotyped virus. f Calu-3 cells transfected with scramble or SENP3-specific siRNAs were infected with SARS-CoV-2 S pseudotyped virus for 48 h, the protein extracts were harvested for luciferase assay. g Calu-3 cells were transfected with scramble or SENP3-specific siRNAs treated with SARS-CoV-2 (MOI = 0.5) for indicated time points. The protein extracts were harvested for immunoblot analysis. h 293 T cells transfected with scramble or SENP3-specific siRNAs were then transfected with plasmids encoding Flag-ACE2 and HA-SUMO3. The protein extracts were harvested for immunoprecipitation and immunoblot analysis. i Luciferase activity of Calu-3 cells transfected with scramble or PIAS4-specific siRNAs, along with SARS-CoV-2 S pseudotyped virus infection for 48 h. j Lysates of Calu-3 cells transfected with scramble or PIAS4-specific siRNAs treated with SARS-CoV-2 (MOI = 0.5) for indicated time points. The protein extracts were harvested for immunoblot analysis. k Lysates of 293 T cells transfected with vectors of HA-SUMO3 and Flag-ACE2 or its K187R mutant along with vector expressing Myc-PIAS4 were immunoprecipitated with anti-Flag and immunoblotted with anti-HA. In a, b, e, f, i, all error bars, mean values ± SEM, P-values are determined by unpaired two-tailed Student’s t-test of n = 3 independent biological experiments. For a–c, g, h, j, k, similar results are obtained by three independent biological experiments. Source data are provided as a Source data file.