SARS-CoV-2 S-protein inhibits ARPE-19 cell proliferation in vitro. A Immunoblot of ACE2 and β-actin in ARPE-19 or HA-ACE2-overexpressed ARPE-19 cells. B Real-time cell analyzer (RTCA) detection of cell proliferation of ARPE-19 cells (wt), S-protein-treated ARPE-19 cells (B), or Flag-S overexpressed ARPE-19 cells (D). C and E Flow cytometry measurement of the cell cycle of ARPE-19 cells (wt), S-protein-treated ARPE-19 cells (+ S-protein), or Flag-S-overexpressed ARPE-19 cells (Flag-S). The black, gray and blank rectangles represent G1, S, and G2 phases respectively. The data represent mean ± SD, n = 3. Unpaired Student’s T-test was used for statistical analysis. *p < 0.05

S-protein induces senescence of ARPE-19 cells. A SA-β-Gal staining of ARPE-19 cells that were treated with media containing PBS (Con) or S-protein for 48 h. B Quantitation of the percentage of SA-β-Gal positive cells per view in A. Ten views were used for quantitation, and the experiment was repeated three times. The data represent mean ± SD, n = 3, ***p < 0.001. C SA-β-Gal staining of the ARPE-19 cells that express empty vector or p3xFlag-S respectively for 48 h. D Quantitation of the percentage of SA-β-Gal positive cells/ total cells/view (a total of 10 views were used for quantitation). The data represent mean ± SD (n = 3), ***p < 0.001. E qPCR to quantitate the expression of cell cycle inhibitors (P53, p21, and p14arf), cytokines (IL-1β, IL-6, IL-8, MCP-1, TGF-β1), MMP3, and iCAM in the cells treated in A and C. F and G ELISA to measure the secretion of IL-1β and IL-6 in ARPE-19 cells that were treated with S-protein for 48 h. H Immunoblot of the expression of p53, p21, Bcl-2, and GAPDH in ARPE-19 cells that were treated with S-protein for 48 h

Knocking down p21 by siRNA reduces S-protein-induced cell senescence. A qPCR to measure the expression of p21 in ARPE-19 cells expressing scramble siRNA or siRNA to P21 followed by treatment with S-protein for 48 h. The data represent mean ± SD, (n = 3), **p < 0.01, ***p < 0.001. B Immunoblot of p21 in ARPE-19 cells that expressed scrambled siRNA or siRNA against p21. C SA-β-Gal stain in ARPE-19 cells that were transfected with scramble or siRNA-P21 followed by treatment with S-protein for 72 h. D Quantitation of the percentage of SA-β-Gal positive cells vs total cell number per view in C. The data represent mean ± SD, (n = 3), **p < 0.01, ***p < 0.001. E SA-β-Gal stain assay, ARPE-19 cells were transfected with empty vector, Flag-S and Flag-S + siRNA-P21 for 72 h. F Quantitation of the percentage of SA-β-Gal positive cells vs total cell numbers per view in E. The data represent mean ± SD, (n = 3), ***p < 0.001

Removing ROS by NAC down-regulates S-protein-induced senescence of ARPE-19 cells. A Flow cytometry assay measuring DCFH-DA-degenerated fluorescence level in ARPE-19 cells that were treated with S-protein for 24 h (upper panel) or transfected with plasmid p3xFlag-S (lower panel). B and C Quantitation of DCFH-DA fluorescence in A. The data represent mean ± SD (n = 3), ***p < 0.001. D SA-β-Gal stain of ARPE cells that were treated with S-protein or S-protein + NAC for 48 h. E Quantitation of the percentage of SA-β-Gal positive cells in D. The data represent mean ± SD (n = 3), ***p < 0.001. F SA-β-Gal stain of ARPE-19 cells that were transfected with p3xflag-S followed by NAC treatment for 48 h. G, Quantitation of percentage of SA-β-Gal positive cells in F. The data represent means ± SD (n = 3), ***p < 0.001. H, immunoblot the expression of P53, P21 and GAPDH in ARPE-19 cells that were treated with media containing PBS (lane 1), S-protein (lane 2) and S-protein + NAC (lane 3)

S-protein induces ER stress in ARPE-19 cells. A and B Immunofluorescence staining of S-protein in S-protein-treated (B) or Flag-S overexpressed ARPE-19 cells (A) that were transfected with pDs-Red-KDEL. C Immunoblot of the expression of CHOP, BIP, Calnexin, ATF3, ATF4, ATF6, and β-actin in ARPE-19 cells treated with S-protein or treated with S-protein S-protein + NAC for 24 h. D Densitometry quantitation of those proteins in C. The data represent mean ± SD (n = 3), *p < 0.05, **p < 0.01

S-protein activates NF-κB pathways in ARPE-19 cells. A Immunofluorescence staining of p65 protein in S-protein-treated or Flag-S-overexpressed ARPE-19 cells. The nuclei were stained with DAPI. The scale bar represents 20 μm. B Cytosol-Nucleus fraction assay to determine the localization of p65 in ARPE-19 cells that were treated with PBS (Con) or S-protein for 48 h. BrgI and GAPDH were used as nuclear and cytosolic markers respectively. C Immunoblot of iκb and GAPDH in ARPE-19 cells that were treated with sham, S-protein and S-protein + NAC respectively. D Densitometry quantitation of iκb vs GAPDH in C. The data represent mean ± SD (n = 3), *p < 0.05. E Immunoblot of the expression of p53, p21 and GAPDH in ARPE-19 cells that were treated with sham, S-protein and S-protein + Bay-11–7082 (NF-κB inhibitor) for 24 h. F Densitometry quantitation of P53 and p21 in E. The P53 and P21 protein level was normalized to GAPDH. The data represent mean ± SD (n = 3), *p < 0.05. G qPCR measuring the expression of p53, p14arf, IL-1β, IL-6, TGF-β1 and iCAM in ARPE-19 cells treated in E. The data represent mean ± SD (n = 3), **p < 0.01, ***p < 0.001

S-protein induces the expression of senescence-associated factors in the zebrafish retina. A H&E analysis of day 2 zebrafish retina post injection of PBS or 6.3 ng S-protein. B Quantitative RT-PCR measuring the expression of P53, P21, P16, IL-1β, IL-6, IL-8, ICAM1, VEGF-A, BIP, CHOP, ATF3, ATF6 in day 2 and day 3 recovered zebrafish retina post injection of S-proteins. The data showed mean ± SD, n = 6. The unpaired student t-test was used for statistical analysis. *p < 0.05, **p < 0.01