Figure 4

ZFP282 is required for breast cancer cell proliferation and tumorigenesis. (a) Depletion of ZFP282 expression. MCF-7 cells were transfected with siRNAs, as indicated or infected with lentiviruses encoding a NS or ZFP282 shRNA. Protein levels were monitored by immunoblot using the indicated antibodies. (b) Proliferation assay. MCF-7 cells transfected with indicated siRNAs were cultured in the absence or presence of 1 nM E2 for 8 days. Cell viability was determined by MTT assay. Data are means±s.d. (n=6). P value was determined by Student’s t-test. (c) Cell cycle analysis. MCF-7 cells transfected with the indicated siRNAs were cultured in the absence or presence of 10 nM E2 for 24 h and subjected to fluorescence-activated cell sorter (FACS) analysis. Data are means±s.d. (n=4). P value was determined by Student’s t-test (*P<0.05). (d–f) Migration, colony formation and invasion assays. MCF-7 cells were infected with lentiviruses encoding a NS or ZFP282 shRNA. Representative images of cell migration, anchorage-dependent colony formation and invasion assays are shown on the left panel of each figure, and the quantitative analysis (n=4, ±s.d.) is shown on the right panel of each figure. Scratch assay was performed on confluent cell monolayers, and the white line indicates the migration front of cells (d). For colony-formation assay, viable colonies were stained with crystal violet; after washing, the dye was extracted with 10% SDS solution and quantified by absorbance at 570 nm (e). Invasion assay was performed using a modified Boyden chamber, as described under Supplementary Information (f). (g) Effect of ZFP282 depletion on the growth of MCF-7 xenograft tumors. MCF-7 cells infected with the indicated lentiviral shRNA vectors were injected subcutaneously in athymic female mice. Tumor growth was monitored for 10 weeks under the treatment with vehicle or E2 in drinking water. Representative images of tumor-bearing mice and their tumors are shown on the left panel and the average (n=4, ±s.d.) was shown on the right panel. P value was determined by Student’s t-test (*P<0.05).