Effects of the stereoisomers of CCG-1423 and related compounds on cell migration and nuclear import of MRTF-A.

<p>(A) Confluent cultures of B16F10 cells were pre-treated with the indicated concentrations of each of the respective compound or vehicle for 12 h, and then they were scratch-wounded with a 20 μl pipette tip. Cell migration was monitored and quantified as described in Materials and Methods. Statistical differences were calculated using student’s t-test. (B and C) B16F10 cells cultured in DMEM-10% serum were pre-treated with 3 μM of the indicated compound or vehicle for 12 h, and then they were restimulated with serum for 15 min (final serum concentration 20%). Vehicle-treated B16F10 cells with or without serum restimulation were used as controls. Cells were stained with anti-MRTF-A antibody (red) and Hoechst (blue). Representative images of control cells and CCG-1423 treated cells are shown (B). The images were analyzed as described in Materials and Methods: nuclear-specific localization [N], diffuse distribution in the nucleus and the cytoplasm [NC], and cytoplasmic localization [C] (C). Each value represents the means ± SEMs of results from three independent experiments. Subcellular localization of MRTF-A was statistically analyzed by a two-way ANOVA. The dose-dependent effects between CCG-1423 <i>S</i> and CCG-1423 <i>R</i> were significantly different (P < 1 × 10⁻⁴). Significance levels were as follows: ^aP < 1 × 10⁻⁴ and ^bP < 1 × 10⁻⁴.