Fig. 6

NKX3.1 and AURKA cross-talk regulates aggressive phenotypes including AR, ARv7 upregulation and AKT activation in CRPC cells. A Phospho-resistant NKX3.1 inhibits cell proliferation more effectively in C4-2 cells as compared to WT NKX3.1. Cell proliferation was measured at indicated times. *P < 0.05, **P < 0.01. B Phospho-resistant NKX3.1 inhibits cell proliferation more effectively in 22Rv1 cells as compared to WT NKX3.1.  *P < 0.05, **P < 0.01. C Ectopic expression of AURKA increases cell proliferation in C4-2 and NKX3.1-C4-2 cells, but not in 3A-NKX3.1-C4-2 cells. AURKA retrovirus was transiently infected in C4-2, NKX3.1 and 3A-NKX3.1 cells and cell growth was measured after 36h using MTT assay. **P < 0.01, and ***P < 0.001. D Colony formation assay showed that 3A-NKX3.1 is more effective in inhibiting colony formation as compared to the WT allele. E Quantitative data analysis of the soft agar experiment from three independent experiments. *P < 0.05. F NKX3.1 and 3A-NKX3.1 fully suppress chemotaxis in C4-2 cells, whereas AURKA knockdown partially suppressed it. The cells were starved in serum-free media for 12 h. Chemotaxis was performed using Boyden chambers. G The plot shows mean ± SEM of cell motility in C4-2, AURKA-knocked down-C4-2, NKX3.1 and 3A-NKX3.1-C4-2 cells from three independent experiments. **P < 0.01. H NKX3.1 and 3A-NKX3.1 fully suppress chemotaxis in 22Rv1 cells, whereas AURKA knockdown partially suppressed it. I Bar graph indicating the extent of migration plotted as mean ± SD of three independent experiments such as the one indicated in H. *P < 0.05. J AURKA overexpression rescues chemotaxis more effectively in C4-2 and NKX3.1-C4-2 cells, as compared to 3A-NKX3.1-C4-2 cells. K Histogram representing the quantification of migration levels, plotted as mean ± SD of three independent experiments. *P < 0.05, **P < 0.01. L Levels of phospho-AKT in NKX3.1 and 3A-NKX3.1 overexpressing C4-2 cells are significantly lower than control cells. Control, NKX3.1-C4-2 and 3A-NKX3.1-C4-2 cells were assayed for p-AKT levels along with AKT and actin. M Quantification of change in AKT phosphorylation levels in response to NKX3.1 and 3A-NKX3.1-expression. Data from three independent experiments was normalized against actin, and represented as mean ± SEM [**P < 0.01, ns not significant]. N Degree of AKT phosphorylation is lowered by ectopic overexpression of wild-type and 3A-NKX3.1 in 22Rv1 cells. O Quantification of AKT phosphorylation levels obtained from three independent experiments such as the one depicted in N. [**P < 0.01, ns not significant]. P WT and 3A-NKX3.1 retroviruses were infected in AURKA overexpressing C4-2 cells and p-AKT levels were analyzed along with AKT and tubulin. Q Data from three independent experiments as in 6P were used for quantification, *P < 0.05, **P < 0.01 relative to control. R AURKA overexpressing 22Rv1 cells were also assessed for p-AKT levels in response to WT and 3A-NKX3.1 overexpression. S Three independent experiments as in 6R were used for quantitative analysis, **P < 0.01. T Both wild-type NKX3.1 and 3A-NKX3.1 deplete AR protein levels in C4-2 cells. U Histogram showing change in AR and NKX3.1 protein levels. Normalized data from three independent experiments, with actin as loading control, was plotted, **P < 0.01 compared to control cells. V Ectopic expression of NKX3.1 and 3A-NKX3.1 depletes AR protein levels in 22Rv1 cells. W Histogram depicting changes in AR protein levels in 22Rv1, NKX3.1-22Rv1 and 3A-NKX3.1-22Rv1 cells. The data from three independent experiments was plotted as mean ± SEM, **P < 0.01 vs 22Rv1 control cells