Figure 4. FHL3 regulated EMT mainly by TGFβ1/Akt/GSK3β/ubiquitin pathway. (A1 and A2) WB assay showed FHL3 knockdown made downregulation of TGFβ1, smad2/3, and smad4 in PANC1_KD1 and BXPC3_KD1 in total protein level. (B1 and B2) WB assay of nucleus-cytoplasm protein showed that FHL3 knockdown hardly changed the expression level of smad2/3 and smad4 in nucleus in PANC1_KD1 and BXPC3_KD1 cells. (C1 and C2) In PANC1_KD1 and BXPC3_KD1 cells, FHL3 knockdown exactly downregulated the absolute and relative expression of phosphorylated AKT (ser473-AKT) more than 50%, p<0.01; and also downregulated the absolute and relative expression of phosphorylated GSK3β (ser9-GSK3β) more than 50%, p<0.01; and upregulated the absolute and relative expression of phosphorylated GSK3β (try216-GSK3β) more than 2-fold, p<0.001. (D1) 0.25μM and 0.50μM GSK3β inhibitor almost eliminated the effect, promoting the TGFβ1/AKT/GSK3β/ubiquitin process, caused by FHL3 knockdown. As treated with GSK3β inhibitor, GSK3β (try216-GSK3β) and E-cadherin were downregulated, snail1 and twist1 were upregulated. (D2) 0.25μM and 0.50μM GSK3β inhibitor reversed the migration ability of PANC1_KD1 and BXPC3_KD1 cells.