CTGF/CCN2 sustains the survival of mammary epithelial cells. (A) HC11-TRE and HC11-TRE-CTGF cells were seeded in serum-free media in the presence of EGF (10 ng/ml) and harvested after 96 hours. Cells were fixed in ethanol and stained with propidium iodide (50 μg/ml) prior to cell cycle analysis by flow cytometry. Data is representative of three determinations. Percentage of total gated cells in each phase of the cell cycle is shown. (B) Graphical analysis of cell cycle distribution by DNA content in HC11-TRE cells or in HC11-TRE-CTGF cells. (C) HC11-TRE and HC11-TRE-CTGF cells were seeded on coverslips in serum-free media in the presence of EGF (10 ng/ml) for 96 hours, then fixed and apoptosis was detected by TUNEL. Left panels depict cell morphology, while the panels on the right display positive TUNEL staining in white. Quantification of TUNEL staining is shown in panel below. (D) HC11-TRE and HC11-TRE-CTGF cells were seeded in serum-free media with EGF (10 ng/ml). Lysates were harvested after 96 hours and the expression levels of the indicated survival proteins was analyzed by western blot. Results are representative of 3 experiments.