Fig. 3

NKX3.1 negatively regulates AURKA’s protein levels by promoting its ubiquitylation. A NKX3.1 overexpression decreases AURKA protein levels in C4-2 cells. B The graph shows statistical analysis of protein levels from three independent experiments normalized to the actin. *P < 0.05, **P < 0.01. C NKX3.1 overexpression decreases AURKA protein levels in 22Rv1 cells. D The graph represents the quantitative analysis of protein levels from three independent experiments. *P < 0.05. E NKX3.1 silencing increases AURKA level in C4-2 cells. F The histogram shows mean ± SD from three independent experiments upon NKX3.1 silencing. *P < 0.05 and ***P < 0.001. G NKX3.1 silencing increases AURKA level in 22Rv1 cells. H Quantitative analysis of AURKA and NKX3.1 protein levels from three independent experiments. Signals are normalized to the actin. *P < 0.05. I NKX3.1 overexpression does not impact AURKA mRNA levels in C4-2 cells, ** P < 0.01. J NKX3.1 knockdown does not change AURKA mRNA levels in C4-2 cells, **P < 0.01. K Silencing of NKX3.1 stabilizes AURKA protein. C4-2 cells were infected with NKX3.1 shRNA lentivirus for 30 h, followed by CHX (20 µg/ml) treatment for 2 and 4 h. L Dot plot showing mean ± SD from three independent experiments upon NKX3.1 silencing. ***P < 0.001. M Silencing of NKX3.1 stabilizes AURKA protein in 22Rv1 cells. N Dot plot depicting mean ± SD from three independent experiments upon NKX3.1 silencing. ***P < 0.001. O NKX3.1 overexpression increases AURKA ubiquitylation in C4-2 and P 22Rv1 cells. Q Ectopic overexpression of S185A-NKX3.1 curtails AURKA protein levels to a greater extent than WT-NKX3.1 as demonstrated by Western blot analysis. R Three independent set of experiments were used for quantification and data plotted as mean ± SEM, **P < 0.01