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. 2020 Nov 4;12(11):3247. doi: 10.3390/cancers12113247

Figure 2.

Figure 2

AURKA negatively regulates SPOP by degrading it in CRPC cells. (A) AURKA negatively regulates SPOP in C4-2 cells. (B) Histogram showing change in SPOP levels with AURKA overexpression. The data are presented as mean ± SEM obtained from three independent experiments. * p < 0.05 vs. C4-2 control cells. (C) AURKA silencing increases SPOP levels in C4-2 cells. C4-2 cells were infected with either scrambled shRNA lentivirus or AURKA shRNA lentivirus, followed by IB. (D) Histogram shows change in SPOP levels with AURKA knockdown. The data are presented as mean ± SEM obtained from three independent experiments. * p < 0.05 vs. C4-2 control cells. (E) Overexpression of AURKA decreased the levels of SPOP protein in 22Rv1 cells. (F) Histogram shows change in SPOP levels with AURKA overexpression. (G) AURKA silencing increases SPOP protein levels in 22Rv1 cells. (H) Histogram shows change in SPOP levels with AURKA knockdown. (I) AURKA overexpression does not affect SPOP mRNA levels. C4-2 cells were infected with AURKA or vector control retrovirus followed by RT-qPCR for mRNA expression. The data are presented as mean ± SEM obtained from three independent experiments. * p < 0.05 vs. C4-2 control cells. (J) AURKA increases SPOP degradation rate in C4-2 cells. C4-2 and AURKA-shRNA-C4-2 cells were treated with cycloheximide (10 μM) for 3 and 6 h and SPOP levels analyzed. The data are presented as mean ± SEM obtained from three independent experiments. * p < 0.05 vs. C4-2 control cells. (K) Graphical representation of SPOP degradation rate in C4-2 cells. (L) AURKA increases SPOP degradation rate in 22Rv1 cells. The data are presented as mean ± SEM obtained from three independent experiments. * p <0.05 vs. 22Rv1 control cells. (M) Graphical representation of SPOP degradation rate in 22Rv1 cells. (N) AURKA degrades SPOP by enhancing its ubiquitylation. C4-2 cells were co-infected with 6x-His-ubiquitin and AURKA retrovirus for 30 h followed by MG132 treatment for 12 h. SPOP was immunoprecipitated and ubiquitylation analyzed using 6x-His antibody. Each experiment was done at least three independent times and representative data are shown.