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×).

CTGF/CCN2 stimulation contributes to HC11 cell proliferation. (A) HC11-TRE and HC11-TRE-CTGF cells were seeded in 96-well microtiter plates in serum-free media with EGF (10 ng/ml). (B) HC11 cells were seeded in serum-free media with EGF (10 ng/ml) plus serum-free conditioned media from HC11-TRE or HC11-TRE-CTGF (1:1 ratio). Cell proliferation was determined at 24, 48, 72, and 96 hours post addition of EGF using the MTT assay. The results are reported as the mean + S.D. of four determinations. *, p < 0.005.

CTGF/CCN2 contributes to the stabilization of surface-level integrin complexes. (A) HC11-TRE and HC11-TRE-CTGF cells were incubated in serum-free media with EGF (10 ng/ml) or identical media containing either an RGD-containing peptide or RAD-containing peptide (500 μM). The MTT assay was performed at 24, 48, 72, and 96 hours. The results are displayed as the mean + the S.D. of four determinations. *, p < 0.005. (B) HC11-TRE and HC11-TRE-CTGF cells were grown in serum free media for 4 days in the presence of EGF (10 ng/ml). Cells were harvested and stained with an anti-β1 integrin antibody, anti-α6 antibody or isotype controls followed by detection with a fluorescence-conjugated secondary antibody and analysis by flow cytometry. The median number of β1 integrin or α6 positive cells is indicated. The data are representative of 4 independent experiments.

CTGF/CCN2 contributes to the formation of focal adhesion complexes in HC11 cells. (A) HC11-TRE and HC11-TRE-CTGF cell protein lysates were used to detect levels of the indicated focal adhesion-related proteins by western blot. (B) HC11-TRE and HC11-TRE-CTGF cells were seeded in 0.5% serum-containing media with EGF (10 ng/ml) for 4 days. The cells were then fixed in paraformaldehyde and stained for the presence of vinculin with Alexaflor488-conjugated secondary antibody and a DAPI nuclear counterstain. In the vinculin panel focal adhesions are shown as punctate bodies primarily near the edges of cells. Magnification = 40×.

CTGF/CCN2 contributes to the epithelial-matrix interaction during lactogenic differentiation in HC11 cells. HC11-TRE and HC11-TRE-CTGF cells were seeded in serum-free conditions on respective coated or uncoated tissue culture dishes and once confluent, induced to differentiate with DIP for 48 hours. Total RNA was harvested and levels of β-casein transcript were determined by northern blot analysis and normalized to actin. Contribution of the matrix proteins to β-casein transcription can be seen by fold induction analysis of the RNA (white bars = HC11-TRE, dark bars = HC11-TRE-CTGF).

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.

CTGF/CCN2 enhances adhesion of HC11 cells through the α6β1 integrin complex. HC11-TRE cells were resuspended in serum-free growth media + EGF (10 ng/ml) and seeded on microtiter wells coated with recombinant CTGF/CCN2 (2 μg/ml) or 1% BSA wells as described in Methods. Adherent cells were fixed , stained, and quantified by reading the absorbance at 570 nm. Data are means of quadruplicate samples, error bars represent standard error. *, ◇, ■, p < 0.005. (A) HC11-TRE cells adhesion in serum-free growth media + EGF (10 ng/ml) with EDTA (2.5 mM), EDTA + Mg2+ (5 mM) or no addition. (B) HC11-TRE cells adhesion in serum-free growth media + EGF (10 ng/ml) with function-blocking antibodies or isotype controls (25 μg/ml).