Lef1 sirna

The short hairpin RNA (shRNA) targeting CCR2 and pGL3-CCR2 lentivirus vector were established by Genepharma (China). Tumor cells were seeded onto six-well plates at 3 × 10⁵ cells per well. When the cell density reached about 70%, lentivirus particles were transfected into cells in the presence of polybrene. Stably transfected cells were selected with 5 μg/ml puromycin and tested regularly by immunoblotting to ensure downregulation or upregulation of CCR2. In some experiments, cells were transiently transfected with β-catenin or Lef-1 siRNA (Genepharma, China) using Lipo3000 (Invitrogen) according to the manufacturer’s instructions.

LEF1 siRNA (si-LEF1), β-catenin siRNA (si-β-catenin) and control siRNA (si-NC) were designed and synthesized by GenePharma (Suzhou, China). The following siRNA sequences were used: si-NC sense (5′–3′) UUCUCCGAACGUGUCACGUTT, si-NC antisense (5′–3′) ACGUGACACGUUCGGAGAATT,si-LEF1 sense (5′–3′) GCUAUCAACCAGAUUCUUGTT, and si-LEF1 antisense (5′–3′) CAAGAAUCUGGUUGAUAGCTT, si-β-catenin sense (5′–3′) GCUUUAGGACUCCACCUUATT,si-β-catenin antisense (5′–3′) UAAGGUGGAGUCCUAAAGCTT. DF1 or ICP2 cells were plated at 1 × 10⁵ cells per well in 12-well plates. After 24 h of culture, the cells were transfected with si-LEF1, si-β-catenin, and si-NC, respectively. At 48 h post-transfection, total RNA and protein were extracted from these transfected cells. The knockdown efficiency of LEF1 and β-catenin was tested by qRT-PCR and Western blot analysis.