2.4. CDH11 Knockdown

For CDH11 loss-of-function studies, we used On-Target plus Human CDH11 siRNA-Smart pool (L-013493-00-0005, Dharmacon, Lafayette, CO, US) that contains 4 siRNA mix. CDH11 #1: 5’-GUGAGAACAUCAUUACUUA-3’. CDH11 #2: 5’-GGACAUGGGUGGACACAUAUG-3’. CDH11 #3: 5’-GGAAAUAGCGCCAAGUUAG-3. CDH11 #4: 5’-CCUUAUGACUCCAUUCAAA-3’. A day before transfection, MDA-MB-231 cell line was seeded into 6-well cell plate at a density of 3 × 10⁵ cells/well in DMEM containing 10% FBS without antibiotics. The Hs578t cell line was seeded in the same condition except the density (2 × 10⁵ cells/well). After 24 hours incubation, 30–40% confluent cells were transfected with siRNA using Lipofectamine 3000 (L3000008, Thermo Fisher Scientific, Waltham, MA, USA) in serum-free DMEM. 80 nM of siRNA was used as final concentration. Six hours after initial transfection, the medium was replaced with fresh DMEM containing 10% FBS. Next day, cells were re-transfected with same siRNA, following same transfection steps. The siRNA-transfected cells were incubated for 48 h since the first transfection and used for western blot, and functional assays.