Figure 2

DDX21 is highly expressed in proliferative breast cancer cell lines and is both nuclear and nucleolar. (A) A panel of 13 different breast cancer cell lines and nontransformed MCF10A cells were analyzed for DDX21 protein expression by western blot analysis. A total of 50 μg of whole cell extracts was used for analysis. Tubulin was used as a loading control. Quantitation of the signal was made by Image J software and normalized by tubulin levels. (B) Equal numbers of the indicated breast cancer cell lines were plated and cell numbers were counted on a daily basis. Bars indicate standard deviation from three separate experiments. Doubling times were estimated based on the growth curves. Doubling time of MDA-MB-231 cells was determined separately. (C) MCF10A cells were cultured in regular DMEM media without insulin and EGF supplements for 48 hours. Cells were then fed with DMEM complete media with either insulin alone, EGF alone or both insulin and EGF together for 24 hours. Cells were then harvested and total cell lysates were subjected to western blot analysis with anti-DDX21 antibody and anti-tubulin antibody. (D) Above-mentioned growth factor-starved MCF10A cells were treated with 100 ng/ml EGF containing complete media for the indicated time points. Whole cell extracts were subjected to western blot analysis with anti-DDX21 antibody and anti-tubulin antibody. Standard error of the mean is indicated with fold-change. ^*, P <0.05. (E) HMECs and breast cancer cell lines were subjected to immunofluorescence analysis with anti-DDX21 to detect cellular localization of DDX21 protein (red). Cell nuclei were demarcated by DAPI (blue) and nucleoli were demarcated by UBF (green). DAPI, 4',6-diamidino-2-phenylindole; DMEM, Dulbecco's modified Eagle's medium; EGF, epithelial growth factor; HMEC, human mammary epithelial cell; UBF, upstream binding factor.