PMC:7321036 / 40229-42525 JSONTXT

{"target":"http://pubannotation.org/docs/sourcedb/PMC/sourceid/7321036","sourcedb":"PMC","sourceid":"7321036","source_url":"https://www.ncbi.nlm.nih.gov/pmc/7321036","text":"To investigate the contribution of the p38/MAPK pathway to cytokine production, SARS-CoV-2-infected ACE2-A549 cells were treated with the p38 inhibitor SB203580. The mRNA of the inflammatory cytokines interleukin-6 (IL-6), tumor necrosis factor alpha (TNF-α), and others increased during infection and were inhibited by p38 inhibition in a dose-dependent manner (Figures 6F, right, and S4A). Interestingly, p38 inhibition also reduced SARS-CoV-2 subgenomic mRNA (Figure 6F, left) in the absence of major cellular toxicity (Figure S5 ), indicative of reduced viral replication. The SB203580-induced decrease in virus production was further confirmed using an anti-SARS-CoV-2 N protein (anti-NP) antibody-based assay (Figure S5, New York Vero E6). Multiplexed ELISA analysis of supernatants of cells from the same experiment demonstrated strong upregulation of inflammatory cytokines at the protein level, including IL-6, CXCL8, CCL20, and CCL2, which were decreased upon p38 inhibition (Figure S4 B; Table S7). However, because SARS-CoV-2 replication is also inhibited by SB203580, we cannot deconvolve the contributions of p38/MAPK pathway activity and SARS-CoV-2 virus presence on cytokine production.\nFigure S5 Pharmacological Profiling for Viral Titers and Cell Viability, Related to Figure 7\nDose response of phosphoproteomics-informed drugs and compounds. Assays performed in New York (N; red, anti-NP; blue TCID50) and Paris (P; red, RT-qPCR; purple, plaque assays) across two cell lines (A549-ACE2 and Vero E6). Cell viability shown in black. Mean of three biological replicates is shown. Error bars are SEM.\nFigure S4 Cytokine Profiling upon Infection, p38 Inhibition, and Cell Cycle Analysis, Related to Figure 6\n(A) RT-qPCR analysis of indicated mRNA from A549-ACE2 cells pre-treated with p38 inhibitor SB203580 at indicated concentrations for one hour prior to infection with SARS-CoV-2 for 24 hours. Statistical test is Student’s t test. Error bars are SD. (B) Same as in (A) but a Luminex-based quantification of indicated cytokines. Error bars are SD. (C) Cell cycle analysis of Vero E6 cells (same as in Figure 6I) upon SARS-CoV-2 infection at an MOI of 1. Cell stained with DAPI DNA stain prior to flow cytometry analysis. Statistical test is Mann-Whitney test. 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