Figure 2.

Inhibition of WNK1 decreases collagen invasion of MDA-MB-231. A, Equal numbers (1 × 10⁴) of control siRNA (siCtrl)- or WNK1 siRNA (siWNK1)-treated MDA-MB-231 cells were seeded on top of the collagen matrices (1.5 mg/mL) and allowed to invade for 48 hours at 37°C. Cells were fixed and stained, and invading cells were counted from each replicate sample (n = 6). The left panel shows a 3D projection reconstructed from the z-stacks of phalloidin/DAPI stained cells (grayscale images) using a confocal microscope at 20× magnification. The bar graph shows quantification of average numbers of invading cells. B, Twenty-four hours after seeding 3 × 10⁴ cells into 3D collagen gels (1.5 or 2.5 mg/mL), cells were treated with DMSO or WNK463 (1 μmol/L) for 24 hours at 37°C. 3D projection image of phalloidin/DAPI stained cells (grayscale images) taken from a confocal microscope at 40× magnification. Graphs represent average numbers of invading cells (n = 3). Dotted red lines indicate cell monolayer. Data are represented as mean ± SD (**, P < 0.01; ***, P < 0.0001, two-tailed unpaired Student t tests).