Effect of TG2 knockdown on mesenchymal markers in A431-P and A431-III cells. (A) The cells were treated with 40 nM of TG2-specific siRNA or control siRNA. At 48 h post-transfection, cell lysates were prepared and subjected to immunoblotting analysis for TG2, Snail fibronectin, N-cadherin, vimentin and β-actin served as internal controls. (B) The cells were plated onto non-fibronectin-coated cover slips in six-well plate for 24 h. The cells were treated with 40 nM of TG2 siRNA or control siRNA, and then immuno-stained for fibronectin (green) and vimentin (red) with the nuclei stained with DAPI (blue). The fluorescence images were visualized using confocal microscopy. (C) Total RNA was extracted at 48 h after siRNA transfection and analyzed for TG2, Snail and MMP-9 by RT-PCR with GAPDH served as the internal control. (D) The culture conditioned media of TG2-silenced cells were collected and normalized by cell numbers prior to gelatin zymography analysis. (E) After TG2 knockdown, a wound healing assay was performed by scratching the cell layer with a pipette tip, and phase-contrast images were taken at 0 h and 24 h later to assess cell migration into the open space. Quantitative data are presented as the mean (± SD) percentage of migration distance (n = 20). * and # indicate a significant difference compared with the respective control (p < 0.05).