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.