Extended Data Fig. 2: Additional biological properties of VIS exerted by a variety of viruses.

a, SA-β-gal staining in human cell lines (RPE1, A549) infected with AAV, lentivirus, HCoV-NL63, and VSV as in Fig. 1g. b, SA-β-gal staining of human primary bronchial or nasal epithelial cells (HBEpC and HNEpC, respectively) infected with HCoV-NL63 and VSV. Mock infected cells as negative control. Quantification of positive cells for n = 3 independent experiments is shown as mean ± s.d.; scale bar = 100 µm. c, Quantification result of SA-β-gal-positive cells (RPE1, A549) after lentiviral infection at MOI as indicated are shown as mean percentage ± s.d. for n = 3 independent experiments. Note that MOI 50 was chosen for VIS induction. d, Quantification result of SA-β-gal-positive RPE1 cells after infection with VSV-ΔG*-CoV-S or VSV-ΔG*-CoV-2-S at MOI as indicated are shown as mean percentage ± s.d. for n = 3 independent experiments. Note that MOI 10 was chosen for VIS induction. e, SA-β-gal staining in human cell lines (RPE1, A549) and primary HNEpC infected with VSV-ΔG*-CoV-S, VSV-ΔG*-CoV-2-S, or VSV-ΔG*/empty vector (VSV-ΔG*-emp) as in Fig. 1g. NIH3T3 as ACE2-negative, infection-resistant control. f, Fluorescence detection of ROS in IMR90 after infection with retrovirus or VSV and treatment with NAC as indicated (upper panels). SA-β-gal staining and quantification of corresponding samples (lower panels). Mock infection or solvent treatment controls (UT) are shown. Representative photomicrographs and quantification are shown as mean percentages ± s.d. for n = 3 independent experiments. Scale bar for ROS and SA-β-gal = 100 µm; for γH2A.X (insets; pink dots reflect foci) 5 µm. g, Quantification of γH2A.X-positive IMR90 cells as in f. n = 3 independent experiments is shown as mean ± s.d. h, 2'3'-cGAMP ELISA analysis of IMR90 infected and treated with GS-441524 as in g. n = 8 independent experiments is shown as mean ± s.d. i, Viral RNA detection in the supernatant of primary nasal epithelial cells with exogenous hACE2 expression (HNEpC-hACE2) infected with SARS-CoV-2, at the indicated time-points. p53 shRNA (shp53) renders cells senescence–incapable, but, unlike treatment with 10 μM GS-441524 (GS), does not block viral replication. Data are shown as mean values + s.d. for n = 3 independent experiments. j, Relative viability of the indicated conditions, each compared to the corresponding untreated (UT) control of SARS-CoV-2-infected or mock-infected cells as in i, 72 h after infection. Data are shown as mean values + s.d. for n = 3 independent experiments. k, GSEA probing selected senescence-related gene sets⁶⁶ by RNA-seq (GSE147507) analysis of NHBE, Calu-3, and A549 cells infected with SARS-CoV-2, compared to corresponding mock-infected controls. Positive NES indicates enrichment in virus-infected cells (dark grey bars), negative NES indicates downregulation in virus-infected cells (light grey bars). NES of FDR q < 0.05 are considered statistically significant and presented (for individual q-values, see Supplementary Information). Biological replicates comprise n = 7 control and n = 3 infected regarding NHBE, n = 3 for each condition regarding Calu-3, and n = 5 control and n = 3 infected regarding A549.