Anti tmprss2

BEAS-2B cells were seeded in 8-well chamber slides (Ibidi, Gräfelfing, Germany). At 80% confluency, cells were treated with nicotine or EC fluids for 24 h in an incubator. Cells were then fixed in 4% paraformaldehyde for 15 min at room temperature and washed several times with DPBS + . Samples were permeabilized with 0.1% Triton X and blocked using 10% donkey serum (Sigma-Aldrich, St. Louis, MO, USA) for 1 h at room temperature, followed by an overnight incubation in primary antibody. After several washes with PBS-T (DPBS + 0.1% Tween), the samples were incubated at room temperature in the dark for 2 h with appropriate secondary antibodies. Samples were washed several times and mounted using Vectashield with DAPI (Vectashield, San Francisco, CA, USA). Fluorescent cells were imaged with a Nikon Eclipse Ti inverted microscope (Nikon Instrument, Melville, NY, USA) using a 60X objective, and images were captured using a high-resolution Andor Zyla VSC-04941 camera (Andor, Belfast, UK). Antibodies used were anti-ACE2 (1:100; R&D System, Minneapolis MN, USA), anti-TMPRSS2 (1:200; Santa Cruz, Dallas, TX, USA). Secondary antibodies were Alexa fluor-488 or Alexa fluor-594 (Thermofisher, Tustin CA, USA).

Atorvastatin (ATS) and Pravastatin (PVS), lipophilic and hydrophilic, respectively, were purchased from Selleckchem and dissolved in sterile DMSO. LPS (Lipopolysaccharides) was purchased from Sigma–Aldrich (St. Louis, MO, USA).
To obtain an adequate inflammatory stimulus, cells were treated for 48 h with ATS or PVS and subsequently stimulated with LPS 1 µg/mL for 4 h before the end of treatment in A549-hACE2 and MRC5 cells, or for 24 h in CaCO2 cells.
Anti-GAPDH, anti-phospho NfKB (Ser-536), and anti-NfKB antibodies were purchased from Cell Signalling Technology (Beverly, MA, USA). Anti-TMPRSS2 and anti-Flotillin-1 were purchased from Santa Cruz Biotechnology (Dallas, TX, USA). Anti-ACE2 antibody and the goat anti-rabbit secondary antibody and goat anti-mouse secondary antibody were purchased from Abcam (Cambridge, UK).

Culture media and chemical compounds were purchased from Life Technologies (Grand Island, NY, USA). Antibodies against phosphor (P)‐EGFR (1:1,000), EGFR (1:1,000), pSTAT3 (1:1,000), STAT3 (1:1,000), pAKT (1:1,000), pERK (1:1,000), IL‐18 (1:200), vimentin (1:1,000), and Ki‐67 (1:1,000) were purchased from Cell Signaling (Temecula, CA, USA). Anti‐AKT (1:1,000), anti‐ERK (1:1,000), anti‐TMPRSS2 (1:500), anti‐AhR (1:500), and anti‐Lamin B (1:1,000) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The antibody against AhR (1:500) was purchased from GeneTex (Irvine, CA, USA). The antibody against β‐actin (1:1,000) and HA (1:1,000) was purchased from Sigma (St. Louis, MO, USA). Alexa Fluor® 488 goat anti‐mouse IgG (1:1,000) was purchased from Life Technologies (Waltham, MA, USA). CH223191 was purchased from Sigma (St. Louis, MO, USA).