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. 2022 Aug 3;40(7):111220. doi: 10.1016/j.celrep.2022.111220

Figure 2.

Figure 2

The NTD modulates the usage of TMPRSS2 and ACE2 for SARS-CoV-2 virus entry

(A) A representative western blot of transfected 293T cell lysates showing spike cleavage in Delta and Kappa and their chimeras. GAPDH was probed as a loading control.

(B) The band intensity from (A) was densitometrically calculated using ImageJ. Total S2-associated spike proteins (S2 and FL) were then normalized against GAPDH across Delta, Kappa, and NTD-bearing chimeras.

(C) The ratio of S2/FL+S2 from the band intensity of S2 and FL shown in (A) was plotted. n ≥ 3.

(D) Delta, Kappa, or chimeras was transduced into either parental 293T cells or 293T cells overexpressing TMPRSS2. The fold increase of the virus entry in T2-overexpressing cells over parental cells is shown above the scatterplots. Mean ± SEM are shown for technical replicates (n = 4; two-sided unpaired Student’s t test).

(E) The entry efficiency of Delta and Kappa in A549-ACE2/TMPRSS2 cells in the presence of TMPRSS2-specific inhibitor camostat or cathepsin-specific inhibitor E64D. The lentivirus pseudotyped with VSV-G was used as a control. The relative light unit (RLU) was normalized with non-drug-added control, giving a percentage of infection. The data showing the SEM from 4 experiments were plotted; the error bars that lie within the datum points are not shown.

(F) The entry efficiency of Delta and Kappa in the presence of camostat in airway organoids.

(G) Delta, Kappa, or chimeras was transduced into either parental 293T cells or 293T cells overexpressing ACE2 with abrogated TMPRSS2 expression (A2delT2). The fold increase of the virus entry in A2delT2 cells over parental cells is shown above the scatterplots. Mean ± SEM are shown for technical replicates (n = 4, two-sided unpaired Student’s t test).

(H) 293T-A2delT2 cells were pretreated with anti-ACE2 antibody before the addition of PV bearing either Delta, Kappa, or chimeras together with virus pseudotyped with VSV-G. The data show the SEM from 2 technical replicates.

In (D) and (F)–(H), data are representative of two experiments. ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001.