Figure 4.

HUWE1-Mediated TIAM1 Degradation Controls HGF-Induced Motility and Invasion

(A) MDCKII cells were transfected with a nontargeting siRNA (Ctrl) or with siRNAs targeting TIAM1 or HUWE1 alone or in combination. Ninety-six hours later, cells were stimulated with 20 ng/ml HGF for 18 hr. Resultant cell scattering was monitored by phase contrast microscopy. Scale bar, 50 μm.

(B) Quantification of (A). Percentage of cell scattering was calculated by counting the percentage of cells with less than three cell-cell adhesions remaining in each colony. At least ten colonies were counted in each of three independent biological replicates. ^∗∗p < 0.001 (unpaired t test).

(C) MDCKII, H1299, H358, and H522 cells were transfected with a nontargeting siRNA (Ctrl) or siRNAs targeting TIAM1 or HUWE1 alone or in combination and seeded in a modified Boyden chamber coated with collagen I to assay for invasion in the presence of 10 ng/ml HGF. After 1 day, invading cells were stained with crystal violet. Panels show representative images from one of at least three independent experiments. Scale bar, 150 μm.

(D–G) Crystal violet from (C) was extracted and absorbance measured at 600 nm. Relative invasion was determined for (D) MDCKII (E) H1299, (F) H358, and (G) H522 cells by relating optical density to a standard curve of the appropriate cells and normalizing this to total cell number for each condition and cell line. Mean values ±SE from three independent experiments. ^∗∗∗∗p < 0.0001 (unpaired t test).

See also Figure S4.