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. 2021 Sep 8;12(1):2214–2227. doi: 10.1080/21505594.2021.1964329

Figure 4.

Figure 4.

4 F restricts apoptosis and inflammatory responses associated with SARS-CoV-2 infection in lung epithelial cells. A-D. Vero E6 and Calu3 cells were uninfected (mock) or infected with SARS-CoV-2 (MOI 0.1) and were treated with media alone (vehicle), or 4 F (10 μM) as in methods. Confluent cells were fixed 48 h post-infection (hpi) followed by processing for flow cytometry. A, B. Flow cytometry was used in Vero E6 cells to determine the percent of viable cells positive for cleaved caspase 3 (a) and the median fluorescence intensity (MFI) of cleaved caspase 3 (b) compared to a negative cell population (shown in light gray). Representative data from three independent experiments are shown. C, D. Flow cytometry was used in Calu3 cells to determine the percent of viable cells positive for cleaved caspase 3 (c) and the MFI of cleaved caspase 3 (d) compared to a negative cell population. Representative data from three independent experiments are shown. E, F. ELISA was used to determine protein levels of IL-6 (ng/ml) secreted by Calu3 (e) and Vero-E6 (f) cells 48 hours post SARS-CoV-2 infection. The compared groups were uninfected cells (mock, light gray), cells infected with SARS-CoV-2 (SARS-CoV-2+, red), and cells infected with SARS-CoV-2 treated with 4 F (light blue). Data represent the mean ± standard error of means (SEM), representing three independent experiments conducted at least in triplicate. The Mann-Whitney was used to compare each group relative to the vehicle control and the p value for this comparison is shown above each column (***p < 0.001). G. Luminex immunoassays were used to determine protein levels of IL-1β, IL-8, IL-10, TNF-α (pg/ml) secreted by Calu-3 cells 48 hours post SARS-CoV-2 infection. The mean value of each measurement (protein levels in cell culture supernatants in pg/ml) was normalized by the mean value of each measurement in the vehicle group and expressed as fold to the mean of the vehicle control group. Data represent the mean ± SEM, representing three independent experiments conducted at least in triplicate. The Kruskal-Wallis statistical test was used to compare 3 groups and the Mann-Whitney was used to compare each group relative to the vehicle control. The p value for this comparison is shown above each column (*p < 0.05, **p < 0.01, ***p < 0.001)