Welfect ex plus transfection reagent

HepG2 cells were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA) and cultured in Dulbecco’s modified Eagle’s medium (DMEM; Hyclone, Logan, UT, USA) containing 10% fetal bovine serum (FBS; Hyclone) and 1% of 100 units/ml of penicillin/streptomycin, and incubated at 37 °C in a humidified 5% CO₂ incubator. Human C1q was obtained from Quidel (San Diego, CA, USA), Collagen I (endotoxin <1.0 EU/ml) from Advanced BioMatrix, Inc. (San Diego, CA, USA), serum free Opti-MEM from Invitrogen (Invitrogen, CA, USA), and WelFect-EX PLUS Transfection Reagent from Welgene (Welgene, Seoul, South Korea). Trizol reagent and the Superscript III kit were obtained from Invitrogen (Invitrogen, CA, USA). Radioimmunoprecipitation (RIPA) buffer, rabbit and mouse secondary antibodies, DDR1 antibody (sc-532), and anti-β-actin antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Total and/or phosphorylated-p38, -JNK, -ERK, -PI3K, -Akt, phosphor-tyrosine, and ERK antibodies were purchased from Cell Signaling. C1q antibody (ab71940) was obtained from Abcam (Cambridge, UK), and anti-Phospho-DDR1 (Y513) Antibody (P00905) from Bosterbio (California, USA). Control and DDR1 siRNA were from Santa Cruz Biotechnology (Santa Cruz, CA), and primers from Genotech (Daejeon, Korea). All other reagents were purchased from Sigma-Aldrich (St. Louis, MO, USA).

Cells were transfected with plasmids at 50% confluence using Welfect-Ex Plus transfection reagent (WelGene, Daegu, South Korea). Briefly, cells were incubated with the transfection complex containing 0.5 μg ARE-luciferase plasmid, 0.05 μg pRLtk control plasmid, and the transfection reagent (2 μg Welfect-Ex and 1.5 μg Enhancer-Q) in serum-and antibiotic-free OptiMEM (Invitrogen, USA). Transfection was continued for 18 h; the cells were then recovered in complete medium for the next 8 h. The cells were treated with 5 and 10 μg/ml of PB for 24 h and were then lysed with lysis buffer [RIPA buffer (Sigma, USA) and protease inhibitor cocktail tablet (Roche, Switzerland)]. Luciferase activities from firefly and Renilla luciferases in total cell lysates were determined using a Dual-Luciferase Assay kit (Promega, Wisconsin, USA) with a 20/20n luminometer (Turner Biosystems, Sunnyvale, CA, USA). Wild-type cells were treated with 5 μM sulforaphane (SFN) for 6 h as a positive control.

Cells were transiently transfected with control vector (pcDNA3.1( + )–6×Myc), IL-32γ vector, or ITGAV vector per well, using WelFect-EX PLUS transfection reagent in OPTI-MEM, according to the manufacturer’s specification (WelGENE, Seoul, Korea)^{7 (link)}. To generate stable cell lines (control vector and IL-32γ-CD133+ A549 cells), transfected CD133+ A549 cells were cultured in G418 containing growth medium (600 μg/ml) for 4 weeks. G418-resistant colonies were expanded and used.