Inhibition of Notch1 abrogates ROCK1 downregulation by MS-275 in HD matrices.A, MTLn3 breast cancer cells invaded into HD matrix for 24 h and were treated with MS-275, the Notch1-1 inhibitor, DAPT, and combined treatments for 48 h. MS-275 alone decreased ROCK1 transcript level but when used in combination with DAPT, this effect was abrogated. Western blotting for Notch1 demonstrated that increased expression of Notch-1 intracellular domain (NICD), following MS-275 treatment. Conversely, ROCK activity was reduced by MS-275 and this effect was lost in cells that were inhibited for Notch-1. B, the knockdown of Notch1 by siRNA demonstrated, there was a reversal of ROCK inhibition by MS-275 to control levels in cells knockdown for Notch1. The activity of ROCK1 was also increased by 1.3 fold when cells were treated with Notch1 siRNA and MS-275 together. Ratio measurements were obtained following normalisation with α–tubulin. Bars indicate standard error from two biological replicates and the experiments were performed thrice for qPCR and twice for Western blot analysis. 1-way ANOVA followed by post-hoc Tukey’s test indicating significant difference at ** p < 0.01.
Raviraj et al. BMC Cell Biology 2012 13:12 doi:10.1186/1471-2121-13-12