Knockdown of Cav1.2 α1c inhibits the attachment and internalization of SARS-CoV-2.
(A) The CACNA1C mRNA level in the indicated siRNA-transfected cells was measured by qPCR. siControl, scrambled siRNA. siCACNA1C, siRNA specific for CACNA1C mRNA. (B) CACNA1C-silenced Vero-E6 cells were infected with HRB25 (M.O.I. = 0.01), and the supernatants were harvested at 24 h post-infection for plaque assays. (C) CACNA1C-silenced cells were incubated with HRB25 (M.O.I. = 10) for 1 h at 4°C. The viral RNA level in the cell lysate was measured by qPCR. (D and E) The expression of ACE2 on the cell surface and in total cells was detected by western blotting (D) and flow cytometry (E) in CACNA1C-silenced Vero-E6 cells. (F) The CACNA1C mRNA level in the indicated siRNA-transfected cells was measured by qPCR. (G) CACNA1C-silenced cells were incubated with HRB25 (M.O.I. = 10) for 1 h at 4°C, and washed with PBS. The viral RNA level in the cell lysate was measured by qPCR. (H) CACNA1C-silenced Vero-E6-ACE2 cells were infected with HRB25 (M.O.I. = 0.01), and the supernatants were harvested at 24 h post-infection for plaque assays. (I and J) CACNA1C-silenced cells were incubated with HRB25 (M.O.I. = 10) at 4°C for 1 h and washed with PBS, then shifted to 37°C for 1 h. The cells were then washed with PBS (I) or acid buffer/trypsin (J). The washed cells were lysed for qPCR to detect SARS-CoV-2 binding to cells (I) or internalized into cells (J). (K) DMSO-, E64D-, camostat mesylate-, or diltiazem-treated CACNA1C-silenced HEK293T cells transiently expressing ACE2 and TMPRSS2 were infected with HRB25 (M.O.I. = 5). The viral RNA level in the cell lysate was measured by qPCR at 6 h post-infection. The data shown are the means ± SDs of three independent experiments or replicates. The two-tailed unpaired Student’s t-test was used for the statistical analysis. ns, not significant, *p < 0.05, **p < 0.01, ***p < 0.001.
