Figure 3.

Cyp24a1 knockout leads to downregulation of the MAPK, PI3K/Akt, TGF-β signaling pathways, and loss of EMT in the BVE^Cyp24a1-null-derived tumor cells. A, Western blot analysis of the p-Erk, p-AKT, and p-Smad2 protein levels in BVE^Cyp24a1-wt-1, BVE^Cyp24a1-wt-2, BVE^Cyp24a1-null-1, and BVE^Cyp24a1-null-2 tumor cell lines. The phosphorylation of these proteins was decreased in both BVE^Cyp24a1-null tumor cell lines. B, Western blot analysis of vimentin, ZEB1, Snail, and E-cadherin expression in BVE^Cyp24a1-wt-1, BVE^Cyp24a1-wt-2, BVE^Cyp24a1-null-1, and BVE^Cyp24a1-null-2 tumor cell lines. Snail and ZEB1 expression was reduced, whereas E-cadherin expression was increased in both BVE^Cyp24a1-null cell lines. The expression of vimentin was not detected. C, Wound-healing assay. Cell migration was measured by the wound-healing assay. Cells were seeded in 6-well plates (10⁵ cells/well) and a linear scratch was created when cells reached confluent monolayer. The cells were further cultured for 16 hours to observe wound healing or cell migration. The wound healing or cell migration is reduced in the BVE^Cyp24a1-null cells. D, Western blot analysis of the CYP24A1, p-Erk, p-AKT, p-Smad2, vimentin, and E-cadherin protein levels in BVE^Cyp24a1-null-1Cyp24a1 tumor cells. Increased phosphorylation of p-Erk, p-AKT, and p-Smad2 and decreased E-cadherin expression are shown in the BVE^Cyp24a1-null-1Cyp24a1 tumor cells following reexpression of CYP24A1.