Blimp1 is expressed in lung cancer cells and its knockdown reduces migration.

<p>(A) Samples of nuclear extracts (20 µg) of A549, H1299, Calu-1, H23 and H441 human lung cancer cells and MCF-7 and MDA-MB-231 (MB-231) breast cancer cells were subjected to immunoblotting for Blimp1 and β-actin, as a control for equal loading. Positions of molecular weight markers are given in the left lane. A representative of two independent experiments with similar results is shown. (B) A549 and (C) H1299 cells were transiently transfected with 10 nM each of <i>siBLIMP1-1</i>, <i>siBLIMP1-2</i> or a scrambled negative control siRNA. Upper panels: Forty-eight h after transfection, WCE (30 µg) were subjected to immunoblotting for Blimp1 and β-actin. The bands were quantified using NIH Image J software and Blimp1 expression normalized to β-actin expression. Normalized Blimp1 expression was determined in two independent experiments and the average values are given below the blots. Lower panels: Alternatively, after 24 h, cultures were trypsinized and 1×10⁵ cells subjected to a migration assay for 16 h, in triplicate. The average migration from three independent experiments ± SD is presented relative to the negative control siRNA (set at 1.0). <i>P</i> values were calculated using Student's <i>t</i>-test. *, <i>P</i><0.005; **, <i>P</i><0.0005. (D) A549 cells were incubated in the presence of 0.5 nM <i>siBlimp1-2</i> or scrambled negative control siRNA for 16 h. Cells were then transfected with Blimp1 expression vector (2 µg per well in 6-well plate) and incubated for 32 h. (Inset) Whole cell lysates (20 µg) were subjected to western blot analysis using antibodies against Blimp1 or β-actin. Cultures were trypsinized and 1×10⁵ cells subjected to a migration assay for 16 h, in triplicate. The average migration from two independent experiments ± SE is presented relative to the negative control siRNA and EV (set at 1.0). Data shown is a representative of two independent experiments with similar results. (E) A549 cells were transiently transfected with 10 nM each of <i>siBLIMP1-1</i>, <i>siBLIMP1-2</i> or a scrambled negative control siRNA. After 48 h, cultures were trypsinized and 1×10⁵ cells subjected to an invasion assay for 16 h, in triplicate. The average data from three independent experiments ± SD is presented relative to the negative control siRNA (set at 1.0). <i>P</i> values were calculated using Student's <i>t</i>-test. *, P<0.01.