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

Figure 7.

Figure 7

AURKA and SPOP are involved in a negative feedback loop in LNCaP cells as well. (A) AURKA negatively regulates SPOP in LNCaP cells. (B) Histogram showing change in SPOP levels with AURKA overexpression. The data are presented as mean ± SEM obtained from three independent experiments. ** p < 0.01 vs. LNCaP control cells. (C) AURKA silencing increases SPOP in LNCaP cells. LNCaP cells were infected with either scrambled shRNA or shRNA targeting AURKA 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 <0.01 vs. LNCaP control cells. (E) Overexpression of SPOP decreased the levels of AURKA in LNCaP cells. (F) Histogram shows change in AURKA levels with SPOP overexpression. The data are presented as mean ± SEM obtained from three independent experiments. ** p < 0.01 vs. LNCaP control cells. (G) SPOP silencing increases AURKA levels in LNCaP cells. (H) Histogram shows change in AURKA levels with SPOP knockdown. The data are presented as mean ± SEM obtained from three independent experiments. ** p < 0.01 vs. LNCaP control cells. (I) AURKA increases SPOP degradation in LNCaP cells. LNCaP and AURKA-LNCaP cells were treated with cycloheximide (10 μM) for 3 and 6 h and SPOP levels analyzed. (J) Graphical representation of SPOP degradation rate in LNCaP cells. The data are presented as mean ± SEM obtained from three independent experiments. * p < 0.05, ** p <0.01 vs. LNCaP control cells. (K) SPOP increases AURKA degradation in LNCaP cells. (L) Graphical representation of AURKA degradation rate in LNCaP cells. The data are presented as mean ± SEM obtained from three independent experiments. * p < 0.05, ** p < 0.01 vs. LNCaP control cells. (M) AURKA degrades SPOP by enhancing its ubiquitylation. LNCaP cells were co-infected with 6x-His-ubiquitin and AURKA retrovirus for 30 h followed by MG132 treatment for 12 h. 6x-His-tagged ubiquitylated proteins were isolated and ubiquitylation analyzed using SPOP antibody. (N) SPOP degrades AURKA via increased ubiquitylation. LNCaP cells were co-infected with 6x-His-ubiquitin and SPOP retrovirus for 30 h followed by MG132 treatment for 12 h. 6x-His-tagged ubiquitylated proteins were isolated and ubiquitylation analyzed using AURKA antibody. Each experiment was done at least three independent times and representative data are shown.