Dapi 4 6 diamidino 2 phenylindole

After treatment with HDM extract or PBS for 24 h, the culture medium was removed, and the MLE-12 cells were washed in PBS 3 times. The cells were then fixed in 4% paraformaldehyde at 4°C for 15 min, followed by 3 washes with PBS. Afterwards, the cells were blocked with 5% goat serum for 1 h at room temperature and were incubated with mouse anti-E-cadherin, rabbit anti- N-cadherin or rabbit anti-SPD primary antibody at 4°C overnight. The cells were washed with PBS 3 times and incubated with Alexa Fluor 555 donkey anti-mouse IgG (H+ L) or Alexa Fluor 647 donkey anti-rabbit IgG (H+L) at 37°C for 1 h in the dark. Next, the cells were washed with PBS and stained with DAPI (4′,6-diamidino-2-phenylindole; Yeasen, China) at 37°C for 10 min in the dark. Images were visualized with a ZEISS LSM710 confocal fluorescence microscope or an Olympus IX73 fluorescence microscope.

MLE-12 cells were seeded in glass-bottom dishes (Thermo, United States) and cultured in a 5% CO₂ atmosphere at 37°C until the cells grew into a single layer. After treatment with PM₂.₅ or PBS for 12 h, MLE-12 cells were washed three times in PBS and then fixed in 4% paraformaldehyde at 4°C for 15 min. After washing three times with PBS, the cells were blocked with 5% goat serum for 1 h at room temperature. Then, the MLE-12 cells were incubated with a rabbit anti-CD146, rabbit anti-SPD, or rabbit anti-AHR primary antibody at 4°C overnight. After washing three times with PBS, the cells were incubated with Alexa Fluor 555 donkey anti-mouse IgG (H + L) or Alexa Fluor 647 donkey anti-rabbit IgG (H + L) at 37°C for 1 h in the dark. Then, the cells were washed three times with PBS and were stained with DAPI (4′,6-diamidino-2-phenylindole; Yeasen, China) at 37°C for 10 min in the dark. Cellular location and quantification of SPD, CD146, or AHR were visualized using a ZEISS LSM710 confocal fluorescence microscope (Zeiss, Jena, Germany) or an Olympus IX73 fluorescence microscope (Olympus, Tokyo, Japan).

SARS-CoV-2 spike (S) expression plasmid was described previously.^{6 (link)} The HA-tagged and GFP-tagged cGAS constructs were described previously.^{66 (link)} DRAQ5 (ab108410) was purchased from Abcam. DAPI (4′,6-diamidino-2-phenylindole) (40728ES03) was from YEASEN. SARS-CoV-2 Neutralizing Antibody (A19215) was from ABclonal. Alexa Fluor 488 Phalloidin (A12379) was from Thermo Scientific. STING-specific inhibitor H-151 (inh-h151) was from InvivoGen. diABZI STING agonist (compound 3) (S8796) and SR-717 lithium (S0853) were from Selleck.