Figure 3. Effects of TOB1 knockdown in MCF7 cells versus LCC1 and LCC9 cells.

A-B. TOB1 knockdown effects on cell proliferation in MCF7, LCC1 and LCC9 cells. 20 nM negative control siRNAs (siNEG) or TOB1 siRNAs (siTOB1) were transfected into cells. After 144 h culture, cells in 96-well plates were stained with crystal violet. A. Representative images of stained cells are shown. B. The cell viability of TOB1 knocked-down cells was quantitated and is presented as average +/− S.D. from 4 independent experiments. Cell viability of siNEG-transfected cells was defined as 100% in each cell line. ** p<0.01 vs. siNEG. C. TOB1 siRNA induces G1/S transition arrest in LCC1 and LCC9, but not in MCF7 cells. 20 nM siNEG or siTOB1 were transfected into cells, and cells were cultured for 48 h and 96 h. BrdU incorporation and flow cytometry were used to measure percentage of replicating cells (numbers shown). 3 independent experiments were repeated. Dot plots shown here were from one typical experiment. D. Cell cycle distribution in siTOB1-transfected cells. Three independent experiments were repeated. Error bar indicates S.D. values. ** p<0.05 vs. siNEG. E. Baseline expression and phosphorylation levels of TOB1 protein in 3 cell lines. Western blots were performed to detect protein levels. Representative images (n=3) of blots are shown. F. Relative protein levels of TOB1 and phospho-TOB1 (Serine 164) were analyzed from 3 independent experiments. Protein level of MCF7 was defined as 1. Error bar indicates S.D. value. * p<0.05 vs. MCF7. G. Relative ratio of phospho-TOB1 (Serine 164) level vs. total TOB1 protein level. The relative ratio of MCF7 was defined as 1. Error bar indicates S.D. value. * p<0.05 vs. MCF7.