L glutamine

Normal human bronchial epithelial (NHBE) cells and human lung cancer cell lines H292, PC-9, CL1-5, H460, H1650, A549, H446, and H1975 were purchased from the Cell Bank of Type Culture Collection of the Chinese Academy of Sciences, Shanghai Institute of Cell Biology. Cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Gibco, CA, USA) supplemented with 10% fetal bovine serum (Gibco), 2 mM l-glutamine (100×; Beyotime, Shanghai, China), 100 U/ml penicillin, and 100 µg/ml streptomycin (100×; Beyotime) and incubated at 37°C in humidified atmosphere containing 5% CO₂.

Oleyl alcohol and dibenzyl ether were purchased from Aldrich Chemical Co. (Saint Louis, MO, USA). Absolute ethyl alcohol was obtained from Chongqing Chuandong Chemical Co. Ltd. (Chongqing, China). Hexane and tetrahydrofuran (THF) were purchased from Adamas Reagent Co. Ltd. (Shanghai, China). DSPE-PEG2000 was obtained from Shanghai Ponsure Biotechnology Co. Ltd. (Shanghai, China). Dulbecco’s Modified Eagle’s Medium (DMEM) was purchased from Life Technologies Co. (Saint Louis, MO, USA). Fetal bovine serum (FBS) was obtained from Zhejiang Tianhang Biotechnology Co. Ltd. (Zhejiang, China). Penicillin/streptomycin solution and L-glutamine were purchased from Beyotime Biotechnology Co. Ltd. (Shanghai, China). Cell Count Kit-8 (CCK-8) was purchased from Boster Biological Technology Co. Ltd. (Wuhan, China). All of these reagents were used as received.

Porcine insulin (28.4 IU/mg) was purchased from Wanbang Biochemical (Jiangsu, China). Phospholipid was obtained from Taiwei Pharmaceutical Co., Ltd. (Shanghai, China). Oleic Acid (OA), Ethyl Oleate (EO), and Isopropyl Myristate (IPM) were purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). Cremophor EL35 (EL35), Transcutol HP and Cremophor RH40 (RH40) were purchased from BASF (Ludwigshafen, Germany). Pepsin, trypsin, 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT), Hank’s balanced salt solution (HBSS), and dimethyl sulfoxide (DMSO) were purchased from Sigma–Aldrich (Steinheim, Germany). Methanol and acetonitrile (HPLC grade) were provided by Tedia Company (USA). penicillin-streptomycin (100 U/mL) and _L-glutamine were purchased from Beyotime Biotechnology (Jiangsu, China). Fetal bovine serum (FBS), 0.25% trypsin-EDTA and Dulbelcco’s Modified Eagle’s Media (DMEM) were obtained from Fisher Scientific France (Illkirch, France). All other reagents employed in this study were of analytical grade.

The rat alveolar macrophage cell line NR8383 (American Type Culture Collection) was maintained in F12 medium (Thermo Fisher Scientific, Inc.) supplemented with 15% fetal bovine serum (Gibco; Thermo Fisher Scientific, Inc.), 100 µg/ml streptomycin, 100 U/ml penicillin and 2 mmol L-glutamine (Beyotime Institute of Biotechnology) at 37°C in a humid atmosphere of 5% CO₂. The medium was discarded and replaced by fresh medium every 3 days until the NR8383 cells reached 60% confluence. Before passaging, adherent cells were harvested, and then centrifuged (100 × g, 4°C, 5 min) and transferred to new microplates.
For induction of ALI in vitro, the cells were stimulated with or without 1 µg/ml LPS for various times (6, 12, 24 and 48 h) at 37°C. Short hairpin (sh)RNA targeting salusin-β and HO-1 together with shRNA control were designed and synthesized by Shanghai GenePharma Co., Ltd. Then, 20 µg pcDNA 3.1 plasmids (Thermo Fisher Scientific, Inc.) and 20 nM shRNAs were transfected into cells at 70–80% confluence using Lipofectamine^® 3000 (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's instructions, as described previously (23 (link)). At 48 h post-transfection, cells were selected for subsequent experiments.