β catenin sirna

SW620 cell line was purchased from ATCC. SW620 cells were cultured with DMEM (#06-1170-87-1A, Biological Industries, Israel) containing 10% fetal bovine serum (#04-011-1A, Biological Industries, Israel), 100 U/ml penicillin and 100 mg/mL streptomycin (#03-034-1B, Biological Industries, Israel). SW620 cells were incubated in a cell incubator (Thermo Scientific, USA) containing 5% CO₂, at 37 °C.
Wnt 5 (#sc-41112) and β-catenin (#sc-29209) siRNA and control (#sc-37007) siRNA were purchased from Santa Cruz Biotechnology (CA, USA). pcDNA-Wnt 5 and pcDNA-β-catenin and control vector were purchased from Addgene (Cambridge, UK). SW620 cells were cultured in DMEM medium at a density of 10⁵ cells in 6-well plates. According to the manufacturer's instructions, lipofectamine 3000 reagent (#L3000015, Invitrogen, CA, USA), pcDNA-Wnt 5 (2 μg), pcDNA-β-catenin (2 μg), control vector (2 μg) or wnt 5 siRNA, β-catenin siRNA (50 nM) and control siRNA (50 nM) were used to transiently transfect into cells. After 6 h of transfection, complete culture medium was added to the transfection medium, continue culturing for 12 h. Then the cells are collected for inoculation.

Akt and β-catenin small interfering RNAs (siRNAs) were designed and synthesized by Shanghai Genepharma (Shanghai, China). Cells were seeded into six-well culture plates at 5 × 105 cells per well before transfected with siRNAs (Akt siRNA, 50 nM; β-catenin siRNA, 50nM) using Lipofectamine2000 (Invitrogen, Carlsbad, CA, USA). About 48 h later, the knockdown efficiency was analyzed by quantitative real-time PCR or western blot. Also, samples treated with siRNAs and leptin were collected and investigated with quantitative real-time PCR or western blot. The tube formation and scratch healing assays were also evaluated. Further, to demonstrate the influence of antagonists on leptin-induced signaling pathways, Wnt pathway inhibitor Dickkopf-1 (Dkk-1, Sigma Aldrich, Darmstadt, Germany) were added to endothelial cells to block the Wnt signaling pathway. After treatment with dkk-1, 5 ng/ml leptin was added to the cells.

β-catenin siRNA and control siRNA were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). pcDNA-β-catenin and control vector were purchased from Addgene (Cambridge, MA, USA). A549 and H1299 cells were cultured in 6-well plates at a density of 10⁵ cells in PRMI1640 medium. After incubation for 12 h, the cells were transiently transfected with pcDNA-β-catenin (2 μg), control vector (2 μg), or β-catenin siRNA (50 nM), and control siRNA (50 nM), using lipofectamine 3000 reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. After transfection for 4–6 h, cells were collected and plated in SFM for another 3 days with or without apatinib.