A549 cell

A549 cells (human LUAD, ATCC®CCL-185™), NCl-H1299 cells (human LUAD, ATCC®CRL-5803™), and HBE cells (human bronchial epithelial cells, ATCC®CRL-2741™) were obtained from ATCC (Manassas, VA, United States). A549 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM, Gibco, Grand Island, NY, United States) containing 10% fetal bovine serum (FBS, Gibco), whereas the culture medium for HBE cells contained 20% FBS. NCl-H1299 cells were cultured in RPMI-1640 medium (Gibco, Grand Island, NY, United States) containing 10% FBS. All the cells were incubated in a humidified incubator at 5% CO₂ and 37°C. The cells that passaged less than five with >70% confluency were used for experiments.

Adenocarcinomic human alveolar basal epithelial cells A549 cells were obtained from ATCC (Manassas, VA, USA). Cells in logarithmic growth phase were digested with trypsin (Gibco/Thermo Fisher Scientific, Waltham, MA, USA) and resuspended in Roswell Park Memorial Institute (RPMI) 1640 medium containing 10% FBS, 100 U/mL penicillin, and 100 μg/mL streptomycin (all from Gibco), and then cultured in a cell incubator with 5% CO₂ at 37 °C. After the cells had adhered to the flask, they were treated with bleomycin (BLM; YZ-1076308), GA (SG8960, both from Solarbio Science & Technology, Beijing, China), or the two combined.

A549 cells (lung adenocarcinoma cells; ATCC, Rockville, MD, USA), which retain the morphological, biochemical and immunological characteristics of type II lung epithelial cells [42 (link)–44 (link)], and Detroit 562 cells (pharyngeal carcinoma derived cells; ATCC, Rockville, MD, USA) are extensively used in investigations of S. pneumoniae adhesion [45 (link)]. The cells were grown in DMEM supplemented with 10% fetal calf serum.

The A549 cells, obtained from ATCC, Manassas, VA, USA, were grown in T75 cell culture flasks in a total volume of 10 mL of Dulbecco’s modified Eagle medium (DMEM) and supplemented with 10% fetal bovine serum, 1% penicillin/streptomycin and kept at 37 °C, 5% CO₂ in humidified air in an incubator.

The A549 cells (human alveolar epithelial adenocarcinoma) were obtained from ATCC (Manassas, VA, USA) and cultured in DMEM/F-12 supplemented with 10% (v/v) FBS and 1% (v/v) antibiotic–antimycotic in a humidified incubator at 37 °C with 5% CO₂. On the day before the addition of treatments, A549 cells were seeded in 96-well plates at a density of 2 × 10⁴ cells/well. Raw FAV and THP, FAV and THP physical mixture, and the spray-dried FAV-THP formulation F1 were dissolved in DMSO and subsequently diluted with complete DMEM/F-12 to concentrations of 1.6–1000 µM. After 24-h incubation with the treatments, the cells were incubated for another 3 h in the MTT solution (0.8 mg/mL). Then, the insoluble formazan was dissolved in IPA, and the absorbance at 570 nm was measured. Cell viability (%) was expressed as the percentage of the absorbance from the cells in the treatments against the absorbance from the cells in the complete DMEM/F-12 with the same concentration of DMSO as the treatment. All groups were repeated three times and each time in triplicates.

Human lung adenocarcinoma A549 cells (Cat # CRL-185, ATCC, Gaithersburg, MD) were grown at 37°C with 5% CO2 in RPMI 1640 (Cat #10-040-CV, Corning, NY, USA) supplemented with 10% fetal bovine serum (FBS) (Cat # FBS-500, X&Y Cell Culture, MI, USA) and 100 units/ml of penicillin/streptomycin (formulated by Norris Comprehensive Cancer Center Media Core, CA, USA). Human AT2 cells were isolated from remnant transplant lung from deceased de-identified non-smoking donors in compliance with USC Institutional Review Board protocol for the use of human source material in research (HS-07-00660). As donors were deceased and de-identified, no patient consent was obtained or necessary. Lungs were processed as previously described [23 (link)]. The three donors were 25, 62, and 67-year-old males who died of non-lung related causes. AT2 cells were isolated from the samples, plated in 50% DMEM/F12 (Cat #D64421, Sigma, MO, USA), 50% DMEM high glucose (Cat #21063, GIBCO, MA, USA), supplemented with 10% FBS, penicillin/streptomycin, 50 ug/ml gentamycin (Cat #G1272, Sigma, MO, USA) and 2.5ug/ml amphotericin (Cat #A2411, Sigma, MO, USA), to allow differentiation to AT1-like cells, and isolated at three different time points (D0, D4, D6) as previously noted [22 (link),23 (link)] (S1 Table).