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. 2017 Aug 1;19(9):672–683. doi: 10.1016/j.neo.2017.06.002

Figure 6.

Figure 6

PTEN was p65 a downstream effector and promoted FBW7 protein degradation and inhibited BC migration.

(A) The indicated cell extracts were subjected to Western blot to determine protein expression of PTEN, AKT, p-AKT(Thr308), and p-AKT(Ser473). (B) The indicated cell extracts were subjected to Western blot to determine protein expression of PTEN. GAPDH was used as a protein loading control. (C) MEF(p65−/−), MEF[p65−/−(p65)] vs. MEF(WT) cells were extracted for total RNA with Trizol reagent. RT-PCR assay was used to determine pten mRNA expression. β-Actin was used as an internal control. (D) Human PTEN promoter-driven luciferase activity was evaluated in MEF(p65−/−), MEF[p65−/−(p65)] and MEF(WT) cells. The results were normalized by internal TK activity. Bars represent mean ± SD from three independent experiments. Student's t test was utilized to determine the P value. The symbol (♣) indicates a significant increase as compared with MEF(WT) cells (♣P < .05), and the asterisk (*) indicates a significant decrease as compared with MEF(p65−/−) cells, *P < .05. (E) Cell lysates and total RNAs extracted from the indicated cells were subjected to either Western blot (top panel) for determination of PTEN, FBW7, and RhoGDIα or RT-PCR (bottom panel) for determination of fbw7 mRNA expression, respectively. (F) The indicated cells were pretreated with proteasome inhibitor MG132 for 10 hours, and the cells were then treated with CHX for the indicated times. Then cell extracts were subjected to Western blot, and GAPDH was used as a protein loading control. (G & H) PTEN overexpression constructs were stably transfected into MEF(WT) cells and T24T cells. The stable cell extracts were subjected to Western blot to determine protein expression of GFP-PTEN, PTEN, FBW7, and RhoGDIα. β-Actin was used as a protein loading control. (I-J) The migration abilities of T24T(GFP-PTEN) and the vector control cells were determined as described in “Materials and Methods” section. Bars represent mean ± SD from three independent experiments. Student's t test was utilized to determine the P value, and the asterisk (*) indicates a significant decrease as compared with vector control cells (*P < .05) (J). (K) The proposed mechanisms underlying p65 overexpression in the promotion of human BC cell migration: p65 overexpression inhibits pten mRNA transcription, which further stabilizes the protein expression of ubiquitin E3 ligase FBW7, in turn increasing the degradation of RhoGDIα protein, finally promoting BC migration.