FIG. 4.

ERK5 regulates iCCA cell migration. (A,B) ERK5 knockdown (shERK5) or nontargeting shRNA‐transfected (shNT) cells were serum deprived for 24 hours and incubated in presence or absence of 10% FBS or 100 ng/mL EGF, as indicated. The number of migrated cells is represented as fold increase over shNT/control. *P ≤ 0.05 vs. shNT/control; **P ≤ 0.05 vs. paired shERK5 (n = 4). (C‐E) Twenty‐four–hour starved iCCA cells were incubated in presence or absence of 100 ng/mL EGF with or without a 30‐minute pretreatment with 5 µM XMD8‐92 or 2 µM AX15836. The number of migrated cells is represented as fold increase over paired control incubated in the absence of EGF. *P ≤ 0.05 versus control. **P ≤ 0.05 versus the relative paired control (n = 4). (F) Lysates from shNT or shERK5‐transfected HuCCT‐1 and CCLP‐1 cells were subjected to immunoblotting with antibodies recognizing ERK5, phosphorylated (p)‐myosin light cain 2 (MLC2), or p‐paxillin. Equal protein loading was assessed by vinculin. Densitometries of p‐MLC2 and p‐paxillin expression (n = 4) are shown in the graphs. *P ≤ 0.05 versus shNT control. Abbreviation: NT, nontargeting.