Figure 2: Six2 regulates stem cell-associated phenotypes in TNBC and mouse mammary carcinoma cells.

A) Six2 knockdown in MDA-MB-231 cells decreases the EpCam⁺/CD44⁺ population, as measured using flow cytometry. Quantification on the right is of triplicate samples for a representative experiment (n=3). Error bars represent the mean +/− SEM. P-values were calculated using one-way anova followed by a Bonferroni multiple comparisons test. B) six2 KD in 66cl4 cells decreases the CD24⁺/CD49f⁺ population, as measured using flow cytometry. Quantification and p-values were done as described above. C) Six2 overexpression in 4T07 cells increases the CD24⁺/CD49f⁺ population, as measured using flow cytometry. Quantification was performed as described above. P-values were calculated using an unpaired two-tailed t test. D) Six2 knockdown in MDA-MB-231 cells decreases tumorsphere formation when compared to control (NT) cells. Quantification on the right is of triplicate samples for a representative experiment (n=3). Error bars represent the mean +/− SEM. P-values were calculated using one-way anova followed by a Bonferroni multiple comparisons test. E) six2 knockdown in 66cl4 cells decreases tumorsphere formation when compared to control (NS) cells. Quantification and p-values were performed as described above. F) Six2 overexpression in 4T07 cells increases tumorsphere formation when compared to control cells. Quantification was performed as described above. P-values were calculated using an unpaired two-tailed t test. G) Six2 knockdown in MDA-MB-231 cells decreases tumor-initiating capacity compared to control (NT) cells. Cells were transplanted into the 4^th mammary fat pad of nude mice at limiting dilutions. The graph displays the percentage of positive tumors at week 5 post tumor cell injection (n=6). Mean tumor-initiating cell (TIC) frequency was determined using the ELDA software program, with estimated ranges of TIC frequency being 72,956–292,560 for NT, 326,264–2,028,088 for KD1 and 175,059–730,575 for KD2.