Ectopic CTGF/CCN2 expression in HC11 cells enhances lactogenic differentiation. (A) HC11-TRE and HC11-TRE-CTGF cells were grown to confluence and exposed to DIP. The cells were photographed at 120 hrs post-DIP addition and are shown at 20× and 40× magnification. The images shown representative of five fields. Mammospheres are bubble-like structures. (B) Mean is the result of the average number of mammospheres in five low power microscopic fields. Data is expressed as the mean + S.D. (C) HC11-TRE and HC11-TRE-CTGF cells were stimulated with DIP for 0, 8, or 16 hours. RNA was extracted and examined by Southern blotting. For quantitation, expression levels of β-casein were normalized to actin and expressed as a fold induction of β-casein mRNA compared to the HC11-TRE control. (D) Chromatin immunoprecipitation analysis of β-casein promoter. HC11-TRE and HC11-TRE-CTGF cells were grown in serum-free media for 24 hours prior to being induced with DIP for 16 hours. (Top) PCR analysis of DNA immunoprecipitated with either an anti-Stat5 antibody or control IgG using primers to amplify the β-casein promotor. (Bottom) Graphical analysis of PCR results from DNA immunoprecipitated with an anti-Stat5 antibody, normalized to the amount of input DNA. Light columns = no DIP, dark columns = DIP (E) CTGF/CCN2 enhances the number and formation of MCF-10A acini in Matrigel. MCF-10A cells were suspended in Matrigel and seeded on Matrigel in 8 well chamber slides. Cells were grown in the absence or presence of CTGF/CCN2 (50 ng/ml) for 20 days. Images in depict acinar structures in culture by phase contrast microscopy at magnificantion 10×. The cells were fixed and the nuclei stained with DAPI. Shown are serial confocal cross sections of acinar structures with respect to the Z axis. The sequential sections reveal the hollow lumen (magnification 20×).