Fig. 7

BMEC infection by SARS-CoV-2 examined after CHIR99021 treatment. A After treatment with CHIR99021 (3 μM) for 1 h, the cells were stained with antibodies against phospho-GYS1 (green) or β-catenin (green). Nuclei were counterstained with DAPI (blue). Nuclear β-catenin was indicated by arrowhead. Bar = 20 μm. B Cytosolic and nuclear fraction of protein lysates were isolated from BMECs treated with CHIR99021. Each protein level of β-catenin was analyzed by western blot. Specificity of the fractionation was confirmed by blotting for β-actin (cytosolic marker) and histone H3 (nuclear marker). Cropped blots were shown and the full-length blots were indicated in Fig. S6. C Cells were treated with CHIR99021 (1–3 μM) 1 h before SARS-CoV-2 infection (MOI = 1). The intracellular viral copy number was determined by RT-qPCR. Normalized value of SARS-CoV-2 RNA copies was indicated against vehicle control as 100%. D Cells were treated with CHIR99021 (3 μM) 1 h before SARS-CoV-2 infection (MOI = 1). Thereafter, viral titers in the culture supernatants were determined by plaque assay in TMPRSS2 expressed Vero E6 cells. Normalized value of SARS-CoV-2 RNA copies was indicated against vehicle control as 100%. Data are represented as mean ± SD (n = 3). *P < 0.05