Six1 overexpression leads to an increase in TICs. (A) Six1 differentially regulates genes in the TIC signature. Microarray analysis was performed on MCF7-Six1 and Ctrl cells . Expression data were filtered for probesets included in the CSC gene list from Shipitsin et al.  that had a 'present' call in at least 50% of the microarrays. Genes were clustered using hierarchical clustering. The color scale represents the expression level of a gene above (red), below (green), and at (black) the mean expression level of that gene across all samples. (B) Graph of the percent CD24low CD44+ cells found in MCF7-Six1 and MCF7-Ctrl cells. The graph represents an average of at least three independent experiments with three individual clones. Error bars represent mean +/- SEM. P values represent statistical analysis using a two-tailed t test. (C) Tumorsphere assays demonstrate that Six1 overexpression increases functional TICs. Secondary tumorsphere assays performed by plating cells from the primary tumorsphere in ultra low attachment plates and culturing for ten days. The graph represents three individual clones. Error bars represent mean value +/- SEM. P values represent statistical analysis using a two-tailed t test. The experiments were performed at least three times. (D) Six1 overexpression in MCF7 cells promotes tumor initiation in NOD/SCID mice. A total of 105 to 103 MCF7-Ctrl or MCF7-Six1 cells were injected into the #4 mammary fat pad of six-week old female NOD/SCID mice, and tumor formation monitored. Data shown from five weeks post injection. Statistical analysis performed using Extreme Limiting Dilution Analysis. Ctrl versus.. Six1; P < 0.001. CSC, cancer stem cell; Ctrl, control; SEM, standard error of the mean; TIC, tumor initiating cell.