Loss of WAVE2 inhibits the oncogenic behavior of TNBC cell lines in vitro. A, Representative WB analysis of protein lysates from MDA-MB-231, MDA-MB-468, and 4T1 breast cancer cell lines and their WAVE2-KO derivatives probed with the indicated antibodies. β-Actin was used as loading control. The numbers under the WB bands represent the fold change of the signal with respect to the CTRL band after normalization to the β-Actin signal. B, Cell proliferation of MDA-MB-231 cells (CTRL) and their WAVE2-deficient derivatives (W2KO) over 3 days. C, Representative micrographs of wound healing assays of confluent cell cultures of MDA-MB-231 (CTRL) cells, their WAVE2-deficient derivatives (W2-KO) that were induced to migrate into scratch wounds in confluent monolayers over 20 hours. Scale bar: 500 μm. D, Quantification of the remaining open wound (open area) at 20 hours from 12 different wounds was measured and plotted as the percentage of the wound at time zero for CTRL cells. E, Representative images of colony formation of the indicated CTRL and W2-KO TNBC cell lines. F, Quantification of the number of colonies. G, Representative micrographs of tumorspheres from CTRL and W2-KO MDA-MB-231. Scale bar: 250 μm for 5x and 100 μm for 10x. Tumorspheres were grown in a 96-well ULA plates and Matrigel (2.5 v/v) was added to the tumorsphere cultures at day 3 and images were captured using Incucyte for 14 days. H, Quantification of the number of tumorspheres. I, Quantification of the number of invading microspheres. Data are the means ± SD (n  =  3; ** and ***, P < 0.01; Student t test). Data shown are representative of three replicates.