Fig. 4.

GSK-3 inhibitor blocks replication in SARS-CoV-2 infected cells: (A) Dose–response analysis of Calu-3 cells treated with GSK-3 inhibitors CHIR99021 or Enzastaurin (University of Pennsylvania). Cells were treated with drug at the indicated concentrations and then inoculated with SARS-CoV-2. Cells were fixed at 48 hpi and total cell count (green) and percent viral infection (blue) detected by immunofluorescence for dsRNA were assessed. (B) Calu-3 cells were treated with vehicle or the indicated concentrations of CHIR99021, inoculated with SARS-CoV-2, fixed at 48 hpi, and Spike protein was detected by immunofluorescence (University of California, Los Angeles). Enzastaurin had no effect on viral infection in Calu-3 cells. Magnification, 100×. (C) Calu-3 cells were treated with vehicle or CHIR99021 (10 µM), inoculated with SARS-CoV-2 at t = 0, and supernatants were sampled at 48 hpi for TCID50 quantification. Median titers are indicated within the boxes (note log₁₀ scale). P = 0.05 (one tailed Mann–Whitney U test). (D) Calu-3 cells were treated with vehicle or CHIR99021, inoculated with SARS-CoV-2 at t = 0, and cell lysates were harvested for immunoblotting for N protein (Upper) or tubulin (Lower) at the indicated times after infection. Abundance of phosphorylated N (slower mobility form) relative to total N protein was assessed densitometrically. Values normalized to 2-h control sample are shown below each lane. A longer exposure of this image is provided in SI Appendix, Fig. S4.