Figure 4

Upregulation of PI3K/Akt-GSK-3β signaling activation is associated with the EMT phenotype in TG2-overexpressing A431-P cells. (A-C) The cells were treated with 25 μM of the specific GSK3 inhibitor SB415286 for 48 h. (A) The cellular protein and RNA levels of Snail and MMP-9 were respectively determined by immunoblotting and RT-PCR. Cyclin D served as the indicator of the inhibition of GSK-3β activity. (B) The secreted MMP-9 activity was detected using gelatin zymography. (C) Cell migratory activity was determined by the wound healing assay. Quantitative data are presented as the mean (± SD) percentage of migration distance (n = 20). (D-F) A431-P cells were transfected with empty pcDNA3.1 vector or pcDNA3.1-TG2, and then treated with 20 μM of PI3K inhibitor LY294002 for 24 h. (D) Cell lysates were analyzed for phosphorylated Akt, GSK-3β, Snail and TG2 using immunoblotting. (E) The secreted activity of MMP-9 was detected by gelatin zymography. (F) Cell migratory activity was determined using the wound healing assay. Quantitative data are presented as the mean (± SD) percentage of migration distance (n = 20). * indicates a significant difference compared with the respective control (p < 0.05). # indicates a significant difference compared with the A431-P (p < 0.05).