Fig. 4: SIX1 promotes breast cancer cells stemness in vitro and tumorigenesis, proliferation, and metastasis in vivo through ZEB1/IL6.

A Western blot analysis of stemness-related proteins in SIX1 overexpressed MCF7, followed by ZEB1 silencing. B Quantitation of Western blot in (A), n = 4. C ALDH activity in Six1-knockdown 66cl4, followed by Zeb1 overexpression, n = 3. D ALDH activity in SIX1 overexpressed MCF7, followed by ZEB1 silencing, n = 3. E Representative images of mammospheres in SIX1 overexpressed MCF7, followed by ZEB1 silencing, scale bar is 100 μm. F Quantitation of mammosphere-formation efficiencies in (E), n = 3. G Quantitation of relative mammosphere size in (E), n = 4. H ELDA assays showed the mammosphere-formation abilities in SIX1 overexpressed MCF7, followed by ZEB1 silencing. I Representative flow cytometry plots of CD24^highCD49f^high subset in Six1-knockdown 66cl4, followed by Zeb1 overexpression. J Quantitation of CD24^highCD49f^high subset in (I), n = 3. K Representative images of tumor formation ability in Six1-knockdown 66cl4, followed by Zeb1 overexpression at three different concentrations, scale bar is 1 cm. L, M Quantitation of tumor formation rate in (K). N Tumor volume of Six1-knockdown 66cl4, followed by Zeb1 overexpression at the end of the limiting dilution experiment. O Representative luminescent images of nude mice in indicated groups taken at the same time point (3 weeks after injection of cells). P Quantitation of luminescent signal of primary tumor in indicated groups, n = 10. Q Kaplan–Meier curve for metastasis-free-survival in indicated groups, n = 10. R Representative luminescent images of organs in indicated groups taken at the same time point (4 weeks after surgery). S HE staining of lung tissue of mice in indicated groups, scale bar is 100 μm. Data were presented as means ± SD; ANOVA or log-rank test were used for statistical analysis. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, and ns P > 0.05. SCR scramble, OE Ctrl overexpressing control, sh#1 #2 two independent shRNA constructs targeting genes, siNT siRNA of negative control, si#1 #2 #3 three independent siRNA constructs targeting genes.