Fig 1: Silencing of RUNX2 or inhibition of the PTEN/Akt signaling pathway neutralizes the protective effect of miR‐149 inhibitor. The PTEN signaling pathway inhibitor SF1670 was added to miR‐149‐overexpressing cells, and RUNX2 was further inhibited in BEWO cells that interfered with miR‐149. (a) The expression of RUNX2, PTEN and Akt1 as well as the extent of Akt1 phosphorylation verified by western blot analysis and RT‐qPCR. (b) Detection of cell proliferation by EdU staining. (c) Cells were stained with CFSE, and cells were tested for cell viability by flow cytometry. (d) Detection of cell apoptosis by flow cytometry. (e and f) Transwell assay detected the migration and invasion of HTR‐8 cells and BEWO cells. Two‐way ANOVA (panel a) or one‐way ANOVA (panel b–f) was adopted for comparisons among multiple groups, followed by Tukey's multiple comparisons test.
Fig 2: miR-320a regulates the PIK3CA/Akt/mTOR signaling pathway in HEH2 cells. Cells were seeded into 6-well plates and transfected with NC-mimics, NC-inhibitor, miR-320a mimics or miR-320a inhibitor for 72 h. α-SMA, collagen I, collagen III, PIK3CA, p-Akt (ser 473), p-Akt (thr 308), Akt, p-mTOR (ser 2481), p-mTOR (ser 2488), mTOR and β-actin protein expression levels were (A) determined by western blotting and (B) semi-quantified. Data are presented as the mean ± SEM from three independent experiments. Comparisons between two groups were analyzed using the unpaired Student's t-test. **P<0.01 vs. NC-mimics; ##P<0.01 vs. NC-inhibitor. miR, microRNA; PIK3CA, phosphoinositide-3-kinase catalytic α polypeptide gene; NC, negative control; α-SMA, α-smooth muscle actin; p, phosphorylated.
Fig 3: Kaplan-Meier survival analysis according to RIOK1 and AKT1 protein expression in patients with glioma. A. Overall survival between the high RIOK1 group and Low RIOK1 group. B. Overall survival between the high AKT1 group and Low AKT1 group.
Fig 4: Silencing RIOK1inhibits glioma cell growth. A. MTT growth curve was used to evaluate cell proliferation ability after transfected with shNC, shRIOK1#1 and shRIOK1#2. B. The results of colony formation assay for U251 and U87 cells. C. The statistical diagram of colony formation assays. **p<0.01, ***p<0.001. D. Transwell invasion assays to evaluate the invasion ability of U87 and U251 cells, the corresponding statistical results are shown below the images.**p<0.01, ***p<0.001. E. Transwell migration assays to assess the migration ability of U87 and U251 cells. F, The protein expression of AKT1 and c-Myc detected by western blot. G. The statistical analysis of western blot gray value results. **p<0.01, ***p<0.001.
Fig 5: The expression of RIOK1 and AKT1 were up-regulated in glioma tissues and correlated with the prognosis of glioma patients. A. Data from TCGA and GTEx (RNA-seq; Normal, n=1152; glioma, n=689) showed that RIOK1/AKT1 were increased in glioma tissues compared with normal brain tissues (*** p<0.001). B. Correlation between RIOK1 and AKT1 expression in TCGA glioma (r=0.440, p<0.001). C. Relative expression of RIOK1 and AKT1 in glioma and normal tissues through IHC. D. RIOK1 and AKT1 expression in Western blot. E. Correlation between RIOK1 and AKT1 expression (r=0.7084, p<0.001). F, G. Relative protein expression of RIOK1 and AKT1 in 6 normal tissues and 30 glioma tissues (*p<0.05, ***p<0,001).
Supplier Page from Abcam for Anti-AKT1 (phospho S473) antibody [EP2109Y] (HRP)