Fig. 5

Cdh1-binding deficient Src mutant promotes breast cancer cell growth. a Immunoblot (IB) analysis of whole-cell lysates (WCL) derived from MDA-MB-231 cells stably expressing empty vector (EV), WT-Src, D-box-1-mutated, or D-box-2-mutated Src. b MDA-MB-231 cells generated in (c) were subjected to cell proliferation assays in DMEM medium supplemented with 10% FBS for 6 days. Cell viability was determined at the indicated time points. The relative cell viability was calculated as mean ± SD (n = 3). *P < 0.05; Student’s t test. c, d MDA-MB-231 cells generated in (a) were seeded for clonogenic survival assays (1000 cells per well). Fourteen days after plating, crystal violet was used to stain the formed colonies (c) and the relative colony numbers were counted as mean ± SD (n = 3), *P < 0.05; Student’s t test (d). e, f MDA-MB-231 cells generated in (a) were subjected to soft agar colony formation assays for 21 days. Formed colonies were stained with iodonitrotetrazolium chloride (INT) (e). The relative colony numbers were calculated as mean ± SD (n = 3). *P < 0.05; Student’s t test (f). g, h MDA-MB-231 cells generated in (a) were subjected to Matrigel invasion assays for 24 h. Invaded cells were stained with phalloidin (g). The invaded distance was calculated as mean ± SD (n = 5). *P < 0.05; Student’s t test (h). i, j MDA-MB-231 cells generated in Supplementary Fig. 5g were seeded for clonogenic survival assays (1000 cells per well). Fourteen days after plating, crystal violet was used to stain the formed colonies (i) and the relative colony numbers were counted as mean ± SD (n = 3), *P < 0.05; Student’s t test (j). k, l Tumor pictures (k) and the growth curves (l) for the xenograft experiments with the MDA-MB-231 cells generated in (a) were inoculated subcutaneously. In each flank of the nude mice, 1 × 10⁶ cells were injected. The visible tumors were measured at the indicated days. Error bars represent ±SEM (n = 4). *P < 0.05; Student’s t test