Figure Legends
FIGURE 1 Codonopsis lanceolata extract and lancemaside A inhibit SARS-CoV-2 infection. (a,b) The effects of 70% EtOH extracts from the aerial parts and roots of Codonopsis lanceolata (CL) on the entry of SARS-CoV-2 pseudovirus into ACE2+ H1299 cells (a) and the viability of H1299 cells (b). (c) The chemical structures of tangshenoside I (1), ethylsyringin (2), syringin (3), sinapaldehyde glucoside (4), lobetyolin (5), lancemasides A (6), B (7), and D (8) obtained from the roots of CL. (d) pSARS-CoV-2 entry assay with eight compounds described in (c) in ACE2+ and ACE2/TMPRSS2+ H1299 cells.
FIGURE 2 Lancemaside A inhibits both endocytic and TMPRSS2-mediated SARS-CoV-2 entry pathways. (a-c) The effects of LA on the entry of SARS-CoV-2 pseudovirus (pSARS-CoV-2) into ACE2+ (a) and ACE2/TMPRSS2+ (b) H1299 cells, and H1299 cell viability (c). The data were representative of three independent experiments with triplicate samples. The error bars indicate the SEM. (d,e) Dose-response curves and confocal images of SARS-CoV-2 N protein (green) and cell nuclei (red) for LA (d) and remdesivir (e) on the infection of ancestral SARS-CoV-2 (black circle) in Vero cells. The blue squares represent Vero cell viability. F Dose-response curve for LA against SARS-CoV-2 recombinant viruses expressing nanoluciferase (Nluc) into A549 cells overexpressing both ACE2 and TMPRSS2. The blue square represents cell viability. The mean ± SEM was calculated from duplicate experiments.
FIGURE 3 Lancemaside A blocks SARS-CoV-2 S-mediated membrane fusion. (a) Schematics for SARS-CoV-2 Spike-ACE2 binding assay using S-RBD-GFP, a recombinant protein comprised of receptor binding domain (RBD) of S protein fused to GFP and ACE2+H1299 cells. (b) The effect of CL extracts and LA on the binding of S-RBD-GFP to ACE2 overexpressed on the surface of H1299 cells was determined by flow cytometry after pretreatment with indicated concentration of CL extracts and LA. The grey peaks indicate the control experiments without S-RBD-GFP addition. The same control data were used for each flow cytometry graphs. (c, d) The effect of LA on membrane fusion between Spike-HEK293 (EGFP+) and ACE2/TMPRSS2+-H1299 (mRuby2+) cells was monitored by time-lapse microscopy (c), and determined by counting the number of EGFP/mRuby2 double positive cells by flow cytometry (d). EGFP+ HEK293 cells (no spike) were used for control experiment. All compounds were used at the concentration of 10 µM. The data were representative of three independent experiments. (c). (f, g) Filipin staining of intracellular cholesterol in ACE2+H1299 and parental H1299 cells after treatment with DMSO or 10 μM LA for 1 h (f) and quantification of the membrane cholesterol levels using Image J (n = 14 for each group). Error bars in the graphs indicate the SEM. P values were determined by the unpaired, two-tailed Student’s t-test. ****P < 0.0001 (g).
FIGURE 4 Lancemaside A inhibits S-mediated syncytia formation.(a) Schematic diagram of the Split-GFP assay to monitor syncytia formation. (b) Experimental timeline for the split-GFP fusion assay. (c) GFP and DAPI (blue) fluorescent images indicate syncytia formed by cell-to-cell fusion and cell nuclei, respectively (upper panel). DAPI nuclear staining overlapped and non-overlapped with GFP fluorescence are pseudocolored in green and red, respectively (lower panel). (d) Quantitative assessment of syncytia formation. GFP and DAPI images were obtained in three random fields per well and the percentage of syncytia formation were calculated by dividing number of nuclei in GFP-positive cells by total number of nuclei. The data were representative of three independent experiments. The error bars indicate the SEM. P values were determined by ANOVA followed by Tukey’s post hoc test. ****P < 0.0001; NS not significant. (e) Western blot analysis using protein lysates from split-GFP experiments (incubation time was 72 h) with anti-cleaved caspase-3 and -9 antibodies. GAPDH was used as a loading control.
FIGURE 5 Lancemaside A inhibits the infection of SARS-CoV-2 WT and D614G mutant. (a) The infection levels of WT and D614G mutant of pSARS-CoV-2 in ACE2+ and ACE2/TMPRSS2+ H1299 cells. P values were determined by the unpaired, two-tailed Student’s t-test (****P < 0.0001). (b) The effects of LA on the infection of WT and D614G mutant of pSARS-CoV-2 in ACE2+ and ACE2/TMPRSS2+ H1299 cells. The data from pSARS-CoV-2 entry assay were representative of three independent experiments with triplicate samples. The error bars indicate the SEM.
FIGURE 6 Lancemaside A effectively inhibits the infection of SARS-CoV-2 variants (a,b) Dose-response curves for LA (a) and remdesivir (b) against various SARS-CoV-2 variants including Alpha (B.1.1.7), Beta (B.1.351), Delta (B.1.617.2), and Omicron (B.1.1.529) in Vero cells. The mean ± SEM was calculated from duplicate experiments. Confocal images of SARS-CoV-2 N protein (green) and cell nuclei (red) at concentrations near the IC50 of LA.