Fig. 2: SARS-CoV-2-infected cells induced paracrine senescence via TNF.
a–e, Early-passage HDFs were cultured for 9 days with culture supernatant of ACE2-HDFs infected with SARS-CoV-2 for 6 days and then analyzed or cultured in plain tissue culture medium for another 5 days. Experimental design (a). Representative photographs of HDFs on day 9 after incubation with culture supernatant of ACE2-HDFs infected with (CoV2-sup.) or without (Mock-sup.) SARS-CoV-2 (b). Relative cell number counted throughout the experiments (c), gene expression analysis on day 9 by RT-qPCR (d) and immunofluorescence staining (e) for indicated genes or proteins on day 9. f–i, Early-passage HDFs were cultured for 9 days with culture supernatant of ACE2-HDFs infected with SARS-CoV-2 for 6 days in the presence or absence of indicated cytokine-inhibitors (TNF-α inhibitor (TNFi), 500 μg ml−1 etanercept; IL-6 inhibitor (IL6i), 50 μg ml−1 tocilizumab; and type I IFN neutralizing antibody (IFNi), 1:200). Experimental design (f). Representative photographs of the cells on day 9 (g). RT-qPCR analysis of day 9 expression of the genes shown at the top of h. Immunoblotting of phospho-p38 and total p38, (left) and relative density of phospho-p38 normalized by total p38 and β-actin using ImageJ (right) (i). Veh, vehicle. β-Actin was used as a loading control. For all graphs, error bars indicate mean ± s.d. of biological triplicate measurements. Statistical significance was determined with a two-tailed unpaired Student’s t test in (d,e) and one-way ANOVA followed by Dunnett’s multiple comparison test (h,i). Scale bars, 50 μm (e), 250 μm (g) and 500 μm (b).
