Nrf2 small interfering rna sirna

The human A549 lung epithelial cell line was selected for in vitro experiment based on a previous study [21 (link)], which was purchased from American Type Culture Collection (ATCC, Manassas, VA, USA), which was cultured in Ham's F-12K medium containing 10% FBS at 37°C in a 5% CO₂ incubator. Compound C was used to inactivate AMPKα, and TAT-14 was used to activate NRF2. The expression of NRF2 in A549 lung epithelial cells was knocked down by NRF2 small interfering RNA (siRNA), which was provided by GenePharma Co. Ltd. (Shanghai, China). On the basis of the manufacturer's instructions, the A549 lung epithelial cells were transfected using Lipofectamine™ 3000 Transfection Reagent (Thermo Fisher Scientific, Waltham, MA, USA). The A549 lung epithelial cells were incubated with LPS at the concentration of 100 μg/mL to establish an in vitro ALI model with or without Kin (15 μM) for 6 h [22 (link), 23 (link)].

3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), endothelial cell medium (ECM), fetal bovine serum (FBS), penicillin/streptomycin, endothelial cell growth factor, and trypsin neutralization solution were purchased from ScienCell (San Diego, California, USA). 0.25% trypsin-EDTA was purchased from Gibco (Grand Island, NY, USA). 2′,7′-Dichlorofluorescein diacetate DCF-DA was purchased from Beyotime. Primary antibodies Nrf2, HO-1, NQO1, GAPDH, and Lamin B1 were from Proteintech (Wuhan, China). Secondary antibodies including anti-mouse and anti-rabbit were purchased from Bioworld Technology (Minnesota, USA). Bardoxolone and ML385 were purchased from MedChemExpress (Shanghai, China). The specific Nrf2 small-interfering RNA (siRNA) and negative control of siRNA were purchased from GenePharma (Shanghai, China). Enhanced chemiluminescence (ECL) western blotting detection solution was obtained from Beyotime (Shanghai, China).

Dulbecco's modified Eagle's Ham/F12 medium (DMEM/F12), trypsin/EDTA, fetal bovine serum (FBS), trypsin/EDTA, antibiotics solution, and sterile phosphate buffer saline were purchased from Invitrogen (Carlsbad, CA). Cell culture dishes were obtained from Corning Inc. (Corning, NY). Taurine, epidermal growth factor (EGF), insulin, hydrocortisone, and other chemicals were supplied by Sigma‐Aldrich (St. Louis, MO). Nrf2 small‐interfering RNA (siRNA) and a negative control (NC) siRNA were designed and synthesized by Genepharma (Shanghai, China). p38 MAPK inhibitor (U0126), JNK inhibitor (Sp600125), and ERK1/2 inhibitor (SB203580) were obtained from Sigma‐Aldrich. The following antibodies were employed in this study: anti‐GRP78, anti‐CHOP, anti‐Nrf2, anti‐HO‐1, anti‐NQO‐1, anti‐Xct, and anti‐Txnrd1 antibodies were purchased from Abcam (Cambridge, UK); anti‐JNK, anti‐P‐JNK anti‐ERK, anti‐P‐ERK, anti‐p38, and anti‐P‐p38 antibodies were purchased from Cell Signaling Technology (Beverly, MA); and anti‐β‐actin antibody was obtained from Amyjet Scientific (Wuhan, China).