Knockdown of pigment epithelium-derived factor expression by siRNA reduces tamoxifen sensitivity in breast cancer cells. (a) siRNA-mediated reduction of pigment epithelium-derived factor (PEDF) expression in MCF-7 and T47D breast cancer cells was performed as described in Materials and methods. Cells were transfected with PEDF or control siRNA for 72 hours and PEDF protein and mRNA levels were determined by western blot and real-time PCR analyses. Transfected cells were also treated with 1 μM 4-hydroxytamoxifen (4OHT) for 72 hours and cell proliferation was determined by DNA quantitation assay and by cell counting using trypan blue exclusion. All experimental points are given as the average of triplicates. (b) Stable expression of PEDF in endocrine-resistant MCF-7:5C and BT474 breast cancer cells sensitized them to tamoxifen. A lentiviral construct encoding the human PEDF cDNA was used to stably express PEDF in MCF-7:5C and BT474 cells. Western blot analysis was used to confirm stable expression of PEDF protein in MCF-7:5C and BT474 cells (top panel). To determine the effect of PEDF expression on tamoxifen sensitivity, lentiviral transduced 5C-PEDF and BT474-PEDF cells were treated with 10-12 M to 10-6 M 4OHT for 72 hours and cell proliferation was determined by DNA quantitation assay (middle panel) and by cell counting using trypan blue exclusion (bottom panel). All experimental points are given as the average of triplicates. Assays were performed twice.