Six1 is dependent on TGF-β and MEK/ERK signaling to increase TICs. (A) Inhibition of TGF-β signaling leads to reduction of the CD24low CD44+ TICs in MCF7-Six1 cells. The graph represents an average of at least three experiments. Error bars represent mean +/- SEM. P values calculated with a two-tailed t test. (B) Inhibition of TGF-β signaling reverses Six1-dependent increases in tumorsphere formation. Representative secondary tumorsphere assay at d10. Experiments were performed at least three times. Error bars represent mean +/- SEM. P values calculated using a two-tailed t test. (C) Six1 expands the MCF7 TIC population through TGF-β signaling. Decreasing cell numbers were injected into the #4 mammary fat pad of six-week old female NOD/SCID mice, and the mice were monitored for tumor formation (five week data shown). Statistical analysis was performed using Extreme Limiting Dilution Analysis: Ctrl versus Six1, P < 5E-08; Ctrl versus TβRIIDN, P = 0.067; Six1 versus TβRIIDN, P < 5E-05. (D) Six1 activates ERK phosphorylation. Western blot analysis performed on lysates from three MCF7-Ctrl and MCF7-Six1 clones using anti-pERK (1:1000) and anti-total ERK (1:1000). (E) Six1 increases ERK phosphorylation in part through TGF-β signaling. The TGF-β type I receptor kinase inhibitor, SB431542, partially represses Six1-mediated ERK phosphorylation. MCF7-Ctrl and MCF7-Six1 cells (three clones each) were treated with 1 μM SB431542 or DMSO for 48 hours after which Western blot analysis was performed and analyzed with Quantity one software (v4.6.2 Bio-Rad). The graph is an average of at least three experiments with three clones. Error bars represent mean +/- SEM. P values calculated using a two-tailed t test. Ctrl, control; ERK, extracellular growth factor receptor; MEK, mitogen activated protein kinase; SEM, standard error of the mean; TGFβ, transforming growth factor β; TIC, tumor initiating cell.