Fig 2: MiR-650 targets to AKT2. *P < 0.05 vs. NC control(A) Luciferase assays. ING4 was used as a positive control. (B–C) Western blot examined AKT2, p-AKT, GSK3β, p-GSK3β, E-cadherin of total protein and β-catenin of nuclear protein. (D) Immunofluorescence of E-cadherin was increased on cell surface in DLD-1 cells and HCT-8 cells by miR-650 (400×).

Fig 3: Correlation between Vav1, miR-29b and Akt2 in MDA-MB-231 cells. (A) qRT-PCR analysis of miR-29b-3p and miR-615-3p in MDA-MB-231 cells in which Vav1 was down-modulated or over-expressed. The values were determined using the 2 −ΔΔCT method and the asterisks indicate statistically significant differences compared to respective controls (Ctrl) taken as 1. (B) Representative immunoblot analysis of Akt2 in lysates from MDA-MB-231 cells transfected with miR-29b-3p inhibitor or mimic. In (C), amounts of Akt2 as deduced from the chemiluminescence signals normalized with β-Tubulin. The asterisks indicate significant differences compared to transfection with scramble oligonucleotides (Negative control, NC), taken as 1. All the data represent the mean of three separate experiments ± SD. * p < 0.05.

Fig 4: Vav1‐dependent regulation of Akt2 in breast cancer‐derived cells with different phenotypes. (A) Representative western blot analysis, performed with the indicated antibodies, of lysates from BT‐474, MCF7, MDA‐MB‐453, and MDA‐MB‐231 cells transfected for 48 h with siRNA specific for Vav1 (Vav1 siRNA) or with a construct expressing human Vav1 (Over Vav1). Scramble siRNA (Ctrl siRNA) and an empty vector were used as controls of the experiment. β‐Tubulin was used as internal control for equivalence of loaded proteins. (B, C) Histograms, as deduced from the densitometry of western blot bands, reporting the levels of proteins normalized to β‐tubulin. All the data are the mean of three separate experiments ±SD. *P < 0.05.

Fig 5: AKT expression in CD133/CD44 sorted cells.A) DLD-1 cells were sorted by flow cytometry and different populations with CD44positive/CD133negative (Q1), CD44positive/CD133positive (Q2), CD44negativeCD133negative (Q3), were collected. B) The sorted cells were further analyzed with western blot for total AKT, AKT1 or AKT2 and betaactin expression.