Biologic Roles of Estrogen Receptor-β and Insulin-Like Growth Factor-2 in Triple-Negative Breast Cancer

Figure 4

Blockade of ligand-induced proliferation of triple-negative MDA-MB-231 cells by ERβ antagonists and by ERβ shRNA. (a) Estrogen receptor-β knockdown. MDA-MB-231 TNBC cells were transfected with shRNA control (CON), ERβ-targeted shRNA (1, 2, 3), and scrambled shRNA vectors (SCRB) (Origene #TG320347). Stable transfectants were selected using puromycin. Cell lysates were processed for Western blot, with results shown for a negative control (CON), 3 different ERβ knockdown clones (1, 2, and 3), and scrambled shRNA (SCRB). β-actin was used as a loading control. (b) Blockade of ligand-induced proliferation of triple-negative MDA-MB-231 cells by ERβ antagonists and by ERβ shRNA. Cells were stably transfected with empty vector (white bars), nonspecific shRNA scrambled-sequence plasmid (grey bars), or an ERβ shRNA plasmid (black bars). As indicated in the figure, cells were treated with different ligands: DPN (ERβ1 agonist), PPT (ERα agonist), and PHTPP (ERβ antagonist) [49, 50]. Cell proliferation was determined after 72 hours using the BrdU cell proliferation ELISA (Roche) (). Graph shows percentage of surviving cells relative to untreated controls (vehicle control), defined as 100% for each transfection condition: empty vector, scrambled, and ERβ shRNA. , , and . Data represents at least three independent experiments.

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