Figure 6

Protein ISGylation in the iPSC-CMs after S-RBD treatment. (a) The mRNA expression levels of IFI6, ISG15, IFITM3, SOCS3, and USP18 were normalized by GAPDH expression and validated using quantitative real-time PCR. ACE2-WT and ACE2-KO iPSC-CMs were treated with 1800 ng/mL S-RBD-sfGFP for 48 h. Relative expression levels were normalized to the expression levels in each iPSC-CM treated with control GFP (n = 5–10 independent biological replicates). Data are presented as medians and interquartile ranges. Statistical differences were calculated using the Kruskal–Wallis test followed by Dunn’s test for multiple comparisons. *p < 0.05, **p < 0.01. (b) The mRNA expression levels of HERC5, HERC6, and EFP were normalized by GAPDH expression and validated using quantitative real-time PCR. ACE2-WT and ACE2-KO iPSC-CMs were treated with 1800 ng/mL S-RBD-sfGFP for 48 h. Relative expression levels were normalized to the expression levels in each iPSC-CM treated with control GFP (n = 5–10 independent biological replicates). Data are presented as medians and interquartile ranges. Statistical differences were calculated using the Kruskal–Wallis test followed by Dunn’s test for multiple comparisons. *p < 0.05. (c) Whole cell lysates were extracted from ACE2-WT-iPSC-CMs and ACE2-KO-iPSC-CMs on day 28 after differentiation and analyzed by western blotting using the indicated antibodies. Both iPSC-CMs were incubated with 1200 ng/mL SARS-CoV-2 S-RBD-sfGFP or control GFP for 48 h prior to western blotting. Original blots are presented in Supplementary Fig. S6. (d) Quantified ISG15 protein expression levels were normalized by GAPDH expression in ACE2-WT-iPSC-CMs and ACE2-KO-iPSC-CMs treated with S-RBD-sfGFP (n = 3–5). Data are presented as medians and interquartile ranges. Statistical differences were calculated using the Mann–Whitney U test. *p < 0.05. (e) Whole cell lysates were extracted from ACE2-WT-iPSC-CMs and ACE2-KO-iPSC-CMs on day 28 after differentiation and analyzed by western blotting using the indicated antibodies. Both iPSC-CMs were incubated with 6000 ng/mL SARS-CoV-2 S-RBD-sfGFP, control-GFP, or 10 IU/mL IFN as a positive control for 48 h before western blotting. Original blots are presented in Supplementary Fig. S6.