Figure 1 | Scientific Reports

Figure 1

From: SARS-CoV-2 spike receptor-binding domain is internalized and promotes protein ISGylation in human induced pluripotent stem cell-derived cardiomyocytes

Figure 1

Internalization of SARS-CoV-2 S-RBD/ACE2 complex into the human iPSC-CMs. (a) Time course of monolayer differentiation into the cardiomyocytes. Differentiated cardiomyocytes were replated on day 14 after differentiation for further analysis. The iPSC-CMs were incubated with purified His-tagged SARS-CoV-2 S-RBD protein for 48 h before immunostaining and western blotting on day 28 after differentiation. (b)Whole cell lysates were extracted from the iPSCs and iPSC-CMs on day 28 after differentiation and analyzed by western blotting using the indicated antibodies. Original blots are presented in Supplementary Fig. S5. (c) The iPSC-CMs were fixed and immunostained with the indicated antibodies on day 28 after differentiation. Nuclei were detected by Hoechst staining. (d) The iPSC-CMs were incubated with 1250 ng/mL purified His-tagged SARS-CoV-2 S-RBD protein for 48 h before immunostaining on day 28 after differentiation with the indicated antibodies. White arrows show the accumulation of S-RBD at the periphery of the iPSC-CMs co-localized with ACE2. (e) The iPSC-CMs were incubated with 1250 ng/mL purified His-tagged SARS-CoV-2 S-RBD protein for 48 h before immunostaining on day 28 after differentiation with the indicated antibodies. Areas enclosed within the white squares are enlarged at the bottom. White arrows indicate the S-RBD/ACE2 complex internalized in the subcellular space. (f) 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. Original blots are presented in Supplementary Fig. S5. (g) Quantified ACE2 protein expression levels were normalized by GAPDH expression in ACE2-WT-iPSC-CMs and ACE2-KO-iPSC-CMs (n = 4). Data are presented as the mean ± SD. Statistical differences were calculated using Student’s t-test. ***p < 0.001. (h) ACE2-WT-iPSC-CMs and ACE2-KO-iPSC-CMs were fixed and immunostained on day 28 after differentiation using the indicated antibodies. (i) ACE2-WT-iPSC-CMs were incubated with 1250 ng/mL purified His-tagged SARS-CoV-2 S-RBD protein for 48 h before immunostaining on day 28 after differentiation with the indicated antibodies. (j) ACE2-KO-iPSC-CMs were incubated with 1250 ng/mL purified His-tagged SARS-CoV-2 S-RBD protein for 48 h before immunostaining on day 28 after differentiation with the indicated antibodies.

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