Fig. 6: I3C inhibited SARS-CoV-2-induced CPE and viral production in Vero E6 cells.
From: Inhibition of HECT E3 ligases as potential therapy for COVID-19
Cells were treated with different doses of I3C (from 50 to 0.069 μM; 1:3 serial dilutions) or DMSO (from 0.5 to 6.9 × 10−4 v/v percentage) 1 h before SARS-CoV-2 infection (MOI = 0.001) in four replicates. Absorption of the virus was allowed for 1 h at 37 °C in presence of I3C or DMSO treatments. The unabsorbed virus was removed and replaced by fresh medium with I3C or DMSO as above. Cells were then treated with either I3C or DMSO every 24 h and incubated at 37 °C with 5% CO2 for 72 h when the survival of infected (A) or not infected (B) cells was measured by crystal violet staining assay. The results were evaluated setting the uninfected control cells as 100% and the remaining values represented as a relative value. Experiments (n = 4) were performed in triplicate and data are expressed as mean +/−SD. Back-titration of virus progeny released by SARS-CoV-2-infected cells, treated as above, was performed on Vero E6 cells. Survival of the cells was measured by crystal violet staining assay. Results were analyzed using Graph Pad (GraphPad Prism 8 XML ProjecT) with nonlinear regression curve fit (Inhibitor vs. response-Variable slope (four parameters)) ((D–F) and data expressed as log TCID50/100 μl (C)). Statistically significant differences between DMSO and I3C are represented as *P < 0.05 or **P < 0.002 determined using the paired t-test. Vero E6 cells were challenged with SARS-CoV-2. After 1.5 d, cells were fixed, stained with an N specific antibody and Hoechst 33342 for cell nuclei. Infected cells and total cell nuclei were counted by image analysis. The infection efficiency was normalized to infection seen in vehicle (DMSO) treated cells (G, H).
