Fig. 6: EMT cells secrete IL6 through the SIX1/ZEB1 signaling to activate the STAT3 pathway and proliferation, migration, and invasion of non-EMT cells.
From: SIX1 transmits signals for breast cancer progression via the ZEB1/IL6/STAT3 signaling axis
A Representative image of the co-culture experiment. B Western blot analysis of STAT3 pathway related protein levels in 66cl4 transfected with shSix1#1 grown for 48 h in indicated CM. C Quantitation of Western blot in (B), n = 3. D Western blot analysis of STAT3 pathway related protein levels in 66cl4 transfected with shSix1#2 grown for 48 h in indicated CM. E Quantitation of Western blot in (D), n = 3. F Western blot analysis of STAT3 pathway related protein levels in MCF7 transfected with OE Ctrl grown for 48 h in indicated CM. G Quantitation of Western blot in (F), n = 3. H Representative IF of p-StaT3 (green) and Stat3 (red) in 66cl4 transfected with shSix1#2 grown for 48 h in indicated CM. DAPI (blue), scale bar is 10 μm. I Quantitation of nuclear/cytoplasmic p-Stat3 fluorescence intensity ratio in (H), n = 4. J Representative IF of p-STAT3 (green) and STAT3 (red) in MCF7 transfected with OE Ctrl grown for 48 h in indicated CM. DAPI (blue), scale bar is 10 μm. K Quantitation of nuclear/cytoplasmic p-STAT3 fluorescence intensity ratio in (J), n = 4. L Representative invasion assay performed on 66cl4 transfected with shSix1#1 grown for 48 h in indicated CM, scale bar is 50 μm. M Quantitation of cell invasion in (L), n = 3. N Representative invasion assay performed on 66cl4 transfected with shSix1#2 grown for 48 h in indicated CM, scale bar is 50 μm. O Quantitation of cell invasion in (N), n = 3. P Representative IF of E-cadherin (red) and N-cadherin (green) of MCF7 transfected with OE Ctrl grown for 48 h in indicated CM with 1 μg/ml IL6 antibody or an equal amount of IgG. DAPI (blue), scale bar is 20 μm. Q, R Quantitation of relative fluorescence intensity in (P), n = 4. S Western blot analysis of Vimentin in 66cl4 transfected with shSix1#1 grown for 48 h in indicated CM. Data were presented as means ± SD; ANOVA were used for statistical analysis. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001. SCR scramble, OE Ctrl overexpressing control, sh#1 #2 two independent shRNA constructs targeting genes, CM conditioned medium.
