Fig. 1: RAC activity is required for BCSCs in vitro and breast tumorigenesis in vivo.

A, B Mammosphere-forming efficiency (%MFE) of human breast cancer cell lines (MCF7, T47D, BT474, and JIMT1) in the presence of RAC-specific inhibitors EHT-1864 (A) and EHop-016 (B) at the indicated concentrations in culture. Values represent the mean ± SD of 3 independent experiments. C Mammosphere-forming efficiency (%MFE) for the secondary mammosphere culture of MCF7 cells, which were treated with the indicated concentrations of EHT-1864 or EHop-016 only during their primary mammosphere culture. Values represent the mean ± SD of 3 independent experiments. D Representative images of structures formed by MCF7 cells that were treated with the indicated concentrations of EHT-1864 or EHop-016 in the primary mammosphere culture either starting from the time of plating (0 h) or 24^th hour after plating. In (A), (B), and (D), the ‘0 uM’ EHop-016 treatment group corresponds to vehicle-only control, which is the same DMSO concentration as in ’10 uM’ treatment group. E Kaplan–Meier graphs for the tumor latency in Rac1^+/+;MMTV-NIC (n = 20), Rac1^flox/+;MMTV-NIC (n = 13), Rac1^flox/flox;MMTV-NIC (n = 2) mice. Time represents postnatal age in days. Median age of palpable tumor formation for each genotype is shown below the graph (*p < 0.05, ***p < 0.01; Lox-rank Mantel Cox test).