Fig. 2

AURKA negatively regulates NKX3.1’s levels by accelerating its ubiquitylation. A AURKA negatively regulates NKX3.1 protein level. AURKA was overexpressed in C4-2 cells, and AURKA and NKX3.1 levels analyzed. B Quantitative analysis of AURKA, NKX3.1 and actin levels upon AURKA overexpression from three independent experiments. The data were normalized to the actin. **P < 0.01. C Overexpression of AURKA decreases NKX3.1 protein level in 22Rv1 cells. D AURKA, NKX3.1 and actin levels were analyzed upon AURKA overexpression from three independent experiments in 22Rv1 cells. *P < 0.05. E AURKA knockdown increases NKX3.1 protein levels. C4-2 cells were infected with AURKA shRNA lentivirus. F Histogram representing the quantitative analysis of detected protein levels from three independent experiments. *P < 0.05, **P < 0.01. G Downregulation of AURKA increases NKX3.1 protein level in 22Rv1 cells. H The graph shows the statistical analysis of the detected protein from three independent experiments. **P < 0.01. I Inhibition of AURKA kinase activity led to elevation of NKX3.1 levels . J Quantification of three independent sets of data represents the extent of increase in NKX3.1 protein levels with inhibition of AURKA using MLN8237 (1 µM for 12 h) in C4-2 cells. **P < 0.01 relative to control. K AURKA overexpression does not alter NKX3.1 mRNA levels in C4-2 and L 22Rv1 cells. The cells were treated with AURKA retrovirus and mRNA levels were analyzed by qRT–PCR from three independent experiments. *P < 0.05, ***P < 0.001. M AURKA silencing did not affect NKX3.1 mRNA level in C4-2 cells and N 22Rv1 cells. The data were normalized to actin. *P < 0.05, ***P < 0.001. O AURKA knockdown stabilizes NKX3.1. C4-2 cells were infected with AURKA shRNA lentivirus following CHX (20 µg/ml) treatment for 2 and 4 h. P Dot plot showing NKX3.1 protein levels from three independent experiments using AURKA shRNA and CHX treated cells as indicated in O. ***P < 0.001. Q AURKA knockdown stabilizes NKX3.1 in 22Rv1 cells. The cells were treated as indicated in O. R The dot plot represents the statistical analysis from three independent experiments of AURKA knockdown in 22Rv1 cells. ***P < 0.001. S Ectopic expression of AURKA results in NKX3.1 ubiquitination in C4-2 and (T) 22Rv1 cells. Cells were infected with AURKA retrovirus and 6x-His-Ub retrovirus for 24 h, and then treated with MG132 for an additional 12 h. NKX3.1 was isolated, and ubiquitylation was detected using a 6x-His antibody