Research Paper Volume 14, Issue 3 pp 1200—1213

Binding of the angiogenic/senescence inducer CCN1/CYR61 to integrin α6β1 drives endocrine resistance in breast cancer cells


Figure 2. CCN1 and D125A-CCN1, but not TM-CCN1, promote acquisition of an endocrine resistance phenotype in MCF-7 breast cancer cells. (A, B) Estradiol (E2)-depleted cells were plated in soft agarose either containing or not E2 (10−9 M), tamoxifen (10−7 M), fulvestrant (10−7 M), their combinations, or vehicles only for either 10 or 18 days. Colony formation (≥50 μm) was assessed using a colony counter. Each experimental value represents the mean colony number (columns) ± S.D. (bars) from at least three separate experiments in which triplicate dishes were counted. (C) Immunoblot analyses of total and activated (phosphorylated) MAPK protein levels in MCF-7/pBABE, MCF-7/CCN1, MCF-7/D125A-CCN1, and MCF-7/TM-CCN1 cells. Blots were reprobed with an antibody for β-actin to control for protein loading and transfer. Results are representative of three independent experiments. (D) Phase contrast images of MCF-7/pBABE, MCF-7/CCN1, MCF-7/D125A-CCN1, and MCF-7/TM-CCN1 cells cultured in Matrigel® in the absence or presence of E2 (10−9 M). Scale bar is 100 μm. 3D cultures were stained for E-cadherin and nuclei were counterstained with DAPI.