Lowered Na+/H+ exchanger regulatory factor 1 (NHERF1) expression leads to increased proliferation in an anchorage-dependent and -independent manner. (a,b) NHERF1 knockdown T47D (a) and MCF7 (b) cells were pulsed with [3H]-thymidine to assay for DNA incorporation. Data are presented as means ± standard error, arbitrarily setting Babe at 100%. The average of three independent experiments was plotted. *P < 0.05, ***P < 0.001. (c) Soft-agar assay. Parental T47D, Babe control, and NHERF-786 knockdown cells (1 × 104) were suspended in 1 ml of 1× culture medium that contained 0.35% agarose. The suspension was added on top of 4 ml of solidified 0.7% agarose. Plated cells were incubated for 20 days at 37°C. Formed colonies larger than 50 μm in diameter were counted. Assays were performed in triplicate. The average of three independent experiments is presented. *P < 0.05, **P < 0.01, in comparison with T47D/NHERF-786.