Rpmi 1640

Primary cultures of bone marrow-derived macrophages (BMDMs) were established from 7–10-week-old C57BL/6J mice, as previously described [29 (link)]. On the 6th culture day, BMDMs were resuspended in L929-conditioned medium (5% L929 supernatant, 10% fetal bovine serum [Gibco, Carlsbad, CA, USA] and 2 mM L-glutamine [Gibco, USA] in RPMI 1640 [CultLab, São Paulo, Brazil]) and seeded at 2.5 × 10⁵ cell density onto 24-well plates. On the next day, BMDMs were washed with PBS and inoculated with 150 µL serum-free RPMI 1640 loaded with either MHV-3 or MHV-A59 at 0.1 MOI. After 1 h of viral adsorption, cells were washed in PBS and cultured for 24 h with 0.5 mL of fresh RPMI 1640 containing calcitriol (Cayman Chemical Co., Ann Arbor, MI, USA) at different concentrations (0.1, 1.0, 2.5, or 5.0 µM) or 0.01% ethanol at 100% (vehicle, corresponding to the 5.0 µM dose of calcitriol). Virus yields were quantified in the supernatant via plaque assay. Cell damage was assessed indirectly by quantifying the abundance/activity of lactate dehydrogenase (LDH) in the supernatant via spectrophotometric measurements (catalog no. K014, Bioclin, Belo Horizonte, Brazil).

Cholesterol efflux assay was performed as previously described [27 (link)]. Briefly, THP-1 monocytes were differentiated with 100 ng/ml phorbol 12-myristate 13-acetate (PMA; Merck) and incubated in serum-containing RPMI-1640 (Thermo Fisher Scientific, USA) with 0.6 μCi/ml [³H]-cholesterol (American Radiolabeled Chemicals Inc., USA) for 72 h. Cells were then incubated for 18 h in serum-free RPMI-1640 containing 4 μM LXR agonist TO-901317 (Cayman Chemical, USA). Cholesterol efflux was performed using 1.1% apoB-depleted plasma for 2 h. ApoB-depleted plasma was obtained after precipitation of apoB-containing lipoproteins with Dextran Sulfate (Merck, USA) as previously described [26 (link)].

NSCLC A549, SPC-A-1, 95D, and NCI-H520 cells and the tunica mucosa bronchiorum epithelium 16HBE cells were obtained from the American Type Culture Collection (Manassas, VA, USA) and maintained in our laboratory. The cells were grown in RPMI 1640 (Gibco, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (Gibco, Grand Island, NY, USA) and 100 units/mL streptomycin/penicillin and cultured at 37°C in a humidified atmosphere with 5% CO₂. For the PD173074 experiments, A549 and A549- pLenti-shRNA1 cells were grown in serum-free and epidermal growth factor (EGF)-free medium (SITA: RPMI 1640 supplemented with 5 µg/mL insulin, 10 µg/mL transferrin, 30 nmol/L sodium selenite, and 0.25% bovine serum albumin) supplemented with PD173074 (dissolved in DMSO, Cayman, USA) at a final concentration of 1 µΜ. The growth media for the control cells were supplemented with equivalent volumes of DMSO without inhibitor.