Fig. 5: The SIX1/ZEB1/IL6 signaling axis activates the STAT3 signaling pathway.

A Representative IF of p-Stat3 (green) and Stat3 (red) in Six1-knockdown 66cl4, followed by IL6 overexpression. DAPI (blue), scale bar is 10 μm. B Quantitation of nuclear/cytoplasmic p-Stat3 fluorescence intensity ratio in (A), n = 4. C Representative IF of p-Stat3 (green) and Stat3 (red) in Six1-knockdown 66cl4, followed by Zeb1 overexpression. DAPI (blue), scale bar is 10 μm. D Quantitation of nuclear/cytoplasmic p-Stat3 fluorescence intensity ratio in (C), n = 4. E Western blot analysis of STAT3 pathway related protein levels in Six1-knockdown 66cl4, followed by IL6 overexpression. F–H Quantitation of Western blot in (E), n = 3. I Western blot analysis of STAT3 pathway related protein levels in Six1-knockdown 66cl4, followed by Zeb1 overexpression. J–L Quantitation of Western blot in (I), n = 3. M Western blot analysis of STAT3 pathway related protein levels in SIX1 overexpressed MCF7, followed by IL6 knockdown. N–P Quantitation of Western blot in (M), n = 3. Q Western blot analysis of STAT3 pathway related protein levels in SIX1 overexpressed MCF7, followed by ZEB1 silencing. R–T Quantitation of Western blot in (Q), n = 3. Data were presented as means ± SD; ANOVA test were used for statistical analysis. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001. SCR scramble, OE Ctrl overexpressing control, shNT shRNA of negative control, sh#1 #2 #3 three independent shRNA constructs targeting genes, siNT siRNA of negative control, si#1 #2 #3 three independent siRNA constructs targeting genes.