Figure 2.

Essential role of Ahnak in EMT-mediated cell migration and invasion. (A,B) HaCaT cells were transfected with the indicated siRNA, serum-starved, and treated with mitomycin C (10 μg/mL), a proliferation inhibitor, for 3 h. Cells were subjected to wound healing assays with or without 5 ng/mL of TGFβ for 24 h and the number of migrated cells (B) was counted. (C,D) HaCaT cells were transfected with the indicated siRNA, serum deprived, and treated with mitomycin C (10 μg/mL) for 3 h. Cells were trypsinized and subjected to Matrigel invasion assays for 48 h in the presence or absence of TGFβ (5 ng/mL). Cells were stained with crystal violet. Invaded cells were observed with an optical microscope (50X). The cell number (D) was counted and graphed as the mean ± SD. of three independent Matrigel invasion assays. Statistical significance was calculated using Student’s t-test. (E) HaCaT cells were transfected with the indicated siRNA, serum deprived, and stimulated with TGFβ (5 ng/mL) for 48 h. Total RNA was isolated, and MMP2 and MMP3 genes expression was quantified by qRT-PCR. The average fold increase, S.D. and statistical significance (P-value from Student’s t-test) were obtained from three independent experiments.