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. Author manuscript; available in PMC: 2018 Feb 1.
Published in final edited form as: J Cell Biochem. 2016 Jun 23;118(2):237–251. doi: 10.1002/jcb.25628

Fig. 7.

Fig. 7

NUMB6 induces Slug expression via activating Akt signaling pathway. (A) Akt1 and Akt2 isoforms phosphorylation was evaluated in DB-7 cells stably expressing control GFP, NUMB4-GFP or NUMB6-GFP by Western blotting. Akt phosphorylation was detected with anti-phospho-AktS473 or AktS474 antibody specific for Akt1 or Akt2 isoform, respectively. Total level of Akt1, Akt2 was determined using anti-Akt1 or anti-Akt2 antibody. β-actin was used as loading controls. (B) Inhibition of Akt phosphorylation reduced Slug expression in NUMB6-GFP DB-7 cells. Western blot analyses were carried out to evaluate the expression of Slug in control vector GFP, NUMB4-GFP or NUMB6-GFP DB-7 cells untreated or treated with 1μM LY294002 inhibitor for 24 hr. NUMB6-induced Slug expression was ablated when cells were treated with LY294002. (C) Control GFP, NUMB4-GFP or NUMB6-GFP DB-7 cells were treated with DMSO or LY294002 for 24 hours. Immunostaining was performed to assess the expression levels of Slug; red is Alexa Fluor-546 for Slug; nuclei were stained with Hoechst 33342. (D) Inhibition of Akt signaling pathway decreased NUMB6-induced DB-7 cell migration and invasion in vitro. In scratch assay confluent monolayers of control GFP, NUMB4-GFP, or NUMB6-GFP DB-7 cells were wounded with a uniform scratch, washed to remove cell debris, and treated with DMSO or 1μM LY294002 inhibitor. Cells were cultured for indicated times in the completed DMEM-F12 medium. Images of cell cultures were captured at 0 and 24 h after scratching. (E) A Matrigel invasion assay was used to characterize FBS-induced invasion of DB-7 cells overexpressing GFP, NUMB4-GFP, or NUMB6-GFP treated with either a vehicle DMSO or LY294002. Cell migration through Matrigel coated membranes is expressed as percentage of the maximal migration induced by 10% FBS. Where indicated, suspended cells were treated with either control vehicle DMSO or 1μM LY294002, placed in migration chambers, and allowed to migrate to FBS for 20 hours. Error bars indicate SEM. Cell invasion for groups treated with LY294002 were compared with DMSO alone as determined using t test. Asterisks indicate significant differences between two groups.