FIG. 6.

Snail is the essential mediator of the EMT induced by the constitutively active IGF-IR signaling. (A) The expression of Snail, Slug, Twist, and Zeb1 mRNA in vector-MCF10A (open bar) and CD8-IGF-IR-MCF10A (shaded bar) cells was examined by Q-PCR. The results are presented as transcript levels relative to the level in vector-MCF10A cells by using the ΔΔCT method, with β-actin mRNA levels used as the normalization control. (B) Vector-MCF10A and CD8-IGF-IR-MCF10A cells were incubated with (+) or without (−) 100 ng/ml IGF-I and 1 μM BMS-536924 for 24 h in growth media. The expression of Snail mRNA was examined by Q-PCR as described in Materials and Methods. The values shown are relative to those of vector-MCF10A cells without IGF-I stimulation. The error bars represent the standard errors of the means. (C) Vector-MCF10A, CD8-IGF-IR-MCF10A, and CD8-IGF-IR/SnaDN-MCF10A cells were grown in their growth media for 2 days. The cells were visualized by phase-contrast microscopy at ×20 magnification (upper panels). The cells were cultured on Matrigel for 8 days, and then the nuclei were labeled with TOPRO-3 and visualized by confocal microscopy with ×40 magnification (lower panels). (D) Vector-MCF10A, CD8-IGF-IR-MCF10A, and CD8-IGF-IR/SnaDN-MCF10A cells were immunoblotted for E-cadherin (E-cad) and Flag. β-Actin was used as a loading control.