Positive association of GSK-3β activity with TG2 and the EMT phenotype in A431-P and A431-III cells. (A) Total cell lysates and cytosolic and nuclear extracts were prepared and analyzed for TG2, NF-κB, and IκBα by immunoblotting. (B) Cells were treated with 25 μM of JSH-23 for 24 h, and the cellular NF-κB of activity was determined using a luciferase reporter assay. (C) The interaction of TG2, NF-κB, and IκBα in A431-P and the A431-III sub-line. (D-E) Cells were treated with 20 or 25 μM of JSH-23 for 24 h, and cell lysates were analyzed for Snail by immunoblotting, and the conditioned media was analyzed for MMP-9 activity by gelatin zymography. (F) Cells were treated with 25 μM of JSH-23 for 24 h, and analyzed for migratory activity using wound healing assay. Quantitative data are presented as the mean (± SD) percentage of migration distance (n = 20). (G) Cells were transfected with control or specific TG2 siRNA, and cellular NF-κB activity was determined using a luciferase reporter assay. * indicates a significant difference compared with the respective control (p < 0.05). # indicates a significant difference compared with the A431-P (p < 0.05). (H) Cellular protein levels of IκBα and TG2 were detected by immunoblotting.