Figure 5. ASCs induce EMT in MCF-7 cells through multiple signaling pathways.

A. The mRNA expression levels of the mitotic marker cyclin B1, transcription suppressor BCL6 and cytokines IL-6, IL-8 and IL-10 were determined by real-time PCR in MCF-7 cells cultured alone or in co-culture with visceral ASCs (ASCvis) from two donors. The results are presented as mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001. Note: each gene expression is demonstrated by ΔCt value, which is normalized to endogenous GAPDH and is reversely related to the amount of target mRNA. B. Western blot analyses with indicated antibodies. Cellular lysates were prepared from MCF-7 control cells and MCF-7 cells indirectly co-cultured with visceral ASCs for 14 days. Lysates from MCF-7 cells exposed to hypoxia (2% O₂) were taken as positive mesenchymal control. β-actin served as loading control. C. Quantification of Western blot analyses in B., relative to corresponding β-actin signal. The results are based on two independent experiments and presented as mean ± SEM. The value in MCF-7 control cells is defined as 1 fold. The quantification was performed with ImageJ. D. MCF-7 cells were cultured alone or with visceral ASCs in the presence of a PI3K inhibitor Wortmannin (50 nM) or a MAPK inhibitor PD98059 (10 μM). After 20 days cellular lysates were prepared for Western blot analyses with indicated antibodies. β-actin served as loading control. E. The treated cells in D. were fixed and stained for the epithelial marker E-cadherin and mesenchymal marker vimentin. Positive cells were counted (n = 200 cells for each condition) and the results are presented as mean ± SD (n = 3).