FIGURE 3.
Functional ER synthesis in infected MDA-MB-231 cells. A, cells were infected with the parent recombinant adenovirus (Ad5) at m.o.i. 150, a recombinant adenovirus bearing cDNA for ERα (α) at 50 m.o.i., ERα203/4/11 (α203/4/11) at 150 m.o.i., or ERα203/4 (α203/4) at 100 m.o.i. In all infections, the total m.o.i. was adjusted to 150 by supplementing with the parent Ad5. Intracellular localization of receptor proteins was examined by ICC. Infected cells as a function of time (shown at 48 h post-infection) were probed with an ERα-specific antibody (HC-20) followed by a fluorescein-conjugated secondary antibody (FITC). 4′, 6-Diamidino-2-phenylindole (DAPI) was used to stain nuclei. B, cell extracts (10 μg) of infected cells at indicated times were subjected to WB using the horseradish peroxidase-conjugated monoclonal FLAG antibody. Molecular mass in kDa is indicated. *72 denotes extracts of the parent adenovirus-infected cells at 72 h. C, cell extracts (10 μg) of infected cells for the indicated times were subjected EMSA in the absence (-) or presence (+) of a FLAG antibody (Ab). *72 indicates extracts of the parent adenovirus-infected cells at 72 h. ERE indicates the unbound radiolabeled ERE, and ER-ERE denotes the radiolabeled ERE-bound ERs. D, effects of ERs on the expression of the C3, TGFA, and TFF1 genes. At 48 h post-infection, cells were treated without (-) or with 10-9 m E2 (E2) for 24 h. Isolated total RNA was processed for and subjected to qPCR. The expression of the genes was compared with that observed in cells infected with Ad5 in the absence of E2, which is set to 1. All experiments are repeated three independent times. Asterisk indicates significant change.