Fig. 3

Cigarette Smoke (CS) extract blocks in vitro SARS-CoV-2 replication in Calu-3 cells. To investigate the effects of cigarette smoke (CS) exposure on SARS-CoV-2 infection, we performed a 72 h in vitro infection of Calu-3 cells, a line permissive to SARS-CoV2 infection and replication. Cells were sham- or CSE-treated for 24 h. Supernatants (SN) and cytoplasmic lysates were obtained from a cell aliquot to measure ACE2 levels by ELISA. Then, cells were infected with SARS-CoV-2 (2 h viral infection in normal media, then remove inoculum). Every 24 h cells were fixed for IF staining of infection, and cell lysates were harvested for SDS-PAGE and WB of viral nucleocapsid (N) protein. dsRNA intermediates arise during the replication of viral RNA (vRNA), and IF staining with dsRNA-specific J2 monoclonal Ab is a good marker for SARS-CoV-2 replication. A Nikon Ti2 automated microscopy was used to quantitatively measure infection, as seen by dsRNA signal. Whereas replication of vRNA peaked at 48 h (A, B) in sham-treated cells, CSE-treatment abrogated infection to levels below the limit of detection. Similar results were seen with WB for viral N protein, showing peak viral protein synthesis at 72 h (C). In C, immunoblots show two bands used for densitometry and separated with a horizontal white line, one for the N protein, and one for GADPH. The two bands were cropped from original gels that are available in a Supplementary repository. ACE2 protein levels were undetectable in the SN, but were unchanged in CSE-treated versus sham cell lysates (not shown). In summary, CSE-pre-exposure increased ACE2 levels but potently abrogated SARS-CoV-2 replication in this in vitro model. The figure is representative of three independent experiments