-repressed HMFs enhance transformation phenotypes of breast cancer cells. (A) HMFs were transfected with either control siRNA or anti-INT6 siRNA. A fraction of these cells was analyzed by Western blot to confirm the reduction in Int6 and α-SMA levels (data not shown). The rest were mixed with MCF7 cells before seeding in triplicate (n = 3). The colonies in each well (a representative area from each is shown below the graph) were counted after 15 days. We note that HMFs seeded alone do not form colonies in soft agar (column 4 and column 5). To confirm that the emerged colonies are of cancer cells, we tagged MCF7 cells with mCherry and found that all colonies were enriched with mCherry-positive cells (right). The HMFs were already tagged by GFP (marked by white arrow heads) , but we did not detect large colonies full of GFP-positive cells. (B) MCF10AT cells were examined similarly as in panel A, except that we did not include the HMF-alone control. (C) SUM102 cells were examined similarly as in panel B. (D) MCF7 cells were loaded in an invasion chamber and submerged in conditioned medium from control or INT6-repressed HMFs. Invaded cancer cells from five different areas (n = 5) on each insert membrane were counted. (E) Normal and INT6-repressed HMFs were mixed with MCF7 cells and seeded in triplicate (n = 3) into soft agar with or without AMD3100 (500 ng/mL). Colonies were counted after 15 days. (F)
INT6-repressed HMFs were co-cultured with MCF7 cells for 4 days before AMD3100 was added (500 ng/mL). After 24 hours, the α-SMA levels in HMFs were measured by Western blot. α-SMA levels, normalized by the loading control GAPDH from the control cells, were set as 1 (n = 4 separate experiments).