Six1 is critical for ERK signaling and for tumor initiation in BALB/c mice. (A) Western blot analysis demonstrates that introduction of a Six1 shRNA leads to an efficient decrease in Six1 protein in the cells. Nuclear extracts were isolated from 66cl4 control and Six1 knockdown cells and western blot analysis was performed with an anti-Six1 antibody (1:1000). An anti-HDAC antibody was used as a loading control. (B) AZD6244 treatment (second lane) and knockdown of Six1 (third and fourth lane) decreases pERK expression in 66cl4 cells. The cells were treated with 10 μM AZD6244 or vehicle for two hours. Whole cell lysates were used for Western blot analysis along with anti-pERK (1:1000) and anti-total ERK (1:1000) antibodies. (C) 66cl4 scramble control or Six1KD cells were serially diluted and then injected into the fourth mammary fat pad of six-week old female BALB/c mice. Mice were monitored for tumor initiation weekly. Data shown are from five weeks post injection of tumor cells. (D) A total of 106 66cl4/scramble or 66cl4/Six1KD cells were injected into the fourth mammary fat pad. One week post injection, mice were treated by oral gavage with vehicle or AZD6244 (50 mg/kg or vehicle twice per day for seven days). After injection of 150 mg of luciferin/kg into the mice, IVIS imaging was used to quantitate the metastastic burden every week. Error bars represent mean value +/- SEM. P values represent statistical analysis between 66cl4/scramble vehicle treated and AZD6244 treated mice, 66cl4/scramble vehicle treated and 66c14/Six1 KD vehicle treated mice, using a two-tailed t test. ERK, extracellular signal-regulated kinase; SEM, standard error of the mean; shRNA, short hairpin RNA.