Figure 1.

Accumulation of β-catenin in MDA-MB-231 cells due to FVIIa treatment. Cells were seeded onto a 12-well plate and allowed to grow. After 2-h serum starvation, cells were treated with FVIIa (100 nm). a, time-dependent accumulation of β-catenin was estimated by standard Western blotting with GAPDH as a loading control. b, band intensity was measured and quantified using ImageJ, and the graph was plotted by GraphPad Prism 5. c, nuclear accumulation of β-catenin was analyzed by Western blotting of nuclear lysate with histone H3 as a loading control. LiCl was used as a positive control. Tubulin was used as a measure of nucleus isolation purity. d, quantification of nuclearly translocated β-catenin was done by ImageJ, and the graph was plotted by GraphPad Prism 5. e, semiquantitative PCR analysis was performed to check the expression status of β-catenin gene in the cells after treatment with FVIIa for various time points. f, quantitative estimation of β-catenin band intensity over GAPDH was done using ImageJ and GraphPad Prism 5. The images of Western blot and RT-PCR analysis are representative of at least three independent experiments. The data are presented as mean ± S.E. Differences are considered to be statistically significant at p < 0.05 using Student's t test. g, cells were grown onto coverglasses followed by treatment with FVIIa. Cells were fixed with 4% paraformaldehyde and permeabilized with 0.01% Triton X-100 followed by immunostaining with β-catenin antibody. DAPI was used for staining nuclei. Scale bars, 25 μm. Quantitative estimation of β-catenin inside the cells and nucleus was performed by ImageJ and MATLAB software; the number of samples (n) per treatment was 23. White arrows indicate nuclear β-catenin. Graphical representations of β-catenin intensity in the nucleus (h) and cells (i) are presented. Error bars represent ±S.E. of the mean. **, p < 0.05; ***, p < 0.001; ns, non-significant using Student's t test; n ≥ 3.