Tle1 specific sirnas

The TLE1 specific siRNAs and the control non-targeting siRNAs were purchased from Invitrogen (Carlsbad, CA). For transient transfection experiments, A549 cells (2×10⁵) were transfected with 25 µM of each siRNA using the Lipofectamine RNAiMAX transfection reagent (Invitrogen). 48 hrs post-transfection, cells were harvested and subjected to immunoblotting or anoikis assays as described below. To generate stable A549 Bit1 knockdown and control pools, A549 parental cells were transfected with pRS vector containing the short hairpin RNA against TLE1 (Origene) or the non-targeting scrambled shRNA (Origene). 48 h post-transfection, 1 µg/ml puromycin (Invitrogen) was added to the medium to select for puromycin-resistant clones. Individual puromycin-resistant clones were screened for Bit1 downregulation by immunoblotting using a specific antibody to Bit11 [15] . Two Bit1 shRNA knockdown-positive clones and two control shRNA clones were pooled for further characterization.