Fig 1: Circ_0016760 silencing blocks NSCLC progression in vivo. BALB/c mice were arbitrarily divided into two groups (n = 5). A total of 2.7 × 106 A549 cells stably expressing Lv-sh-NC or Lv-sh-circ_0016760 were subcutaneously injected into the mice. (a) The representative images of tumors in the Lv-sh-NC group and the Lv-sh-circ_0016760 group are shown. (b) Tumor volume was recorded every 4 days as length × width2 × 0.5. (c) Tumors were weighed after 28 days of inoculation. (d and e) The expression of circ_0016760 and miR-646 was determined by qRT-PCR. (f) Western blot assay was implemented to measure the protein level of AKT3 in tumor tissues. *p < 0.05, **p < 0.01
Fig 2: Inhibition of AKT3 and RAC1 enhances the inhibitory effect of BP on osteoclasts. (A) THP-1 cells were treated with pamidronate for 24 h, followed by cell viability assay using CCK-8 reagent. (B) THP-1 cells were transfected with AKT3 or RAC1 siRNA and the cells were harvested for western blotting analysis 48 h after transfection. (C) Effect of AKT3 or RAC1 siRNA on osteoclast formation. (D) Number of nuclei of multinuclear osteoclasts was counted and the rate of multinuclear osteoclasts was calculated. The apoptosis of THP-1 cells was (E) detected by flow cytometry and (F) quantified. Data are presented as the mean ± SD. The experiments were repeated three times. *P<0.05, **P<0.01. Magnification, x200. BP, bisphosphonates; NC, negative control; si, small interfering; Con, control; sRANKL, soluble receptor activator of NF-?B ligand; M-CSF, macrophage-colony-stimulating factor.
Fig 3: Identification of the interaction between miR-424-5p and AKT3 mRNA using dual luciferase reporter assay. Dual luciferase assay was performed in 293T cells transfected with reporter vectors with wild type or mutant 3'-UTR sequences along with agomiR-negative control (NC) or agomiR-424-5p. The fluorescence values of each group of cells were measured using renilla fluorescence activity as an internal reference. Data are reported as means±SD. *P<0.05 compared with the NC group (ANOVA).
Fig 4: Modulation of Akt isoforms altered E-cadherin-mediated chemoresistance. (A) Western blot validation of Akt isoform expression levels in control or Akt isoform shRNA stable expressing DU-H cells. (B) Western blot of p-Akt and pan-Akt in Akt isoform knockdown and control DU-H cells upon camptothecin (CPT) + tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) treatment for the indicated times. Glyceraldehyde 3-phosphate dehydrogenase was used as the loading control. (C) Immunofluorescence of c-casp3 (red) in Akt isoforms knockdown DU-H cells treated with CPT + TRAIL for 4 h. Bar = 100 µm. (D) Enumeration of c-casp3+ cells with 4 fields per slide, using Student’s t-test, *P < 0.05; ***P < 0.001. (E) Western blot of DU-L cells transiently transfected with GFP or Akts-GFP plasmids for 48 h. Akt1, Akt2, Akt3, and GFP antibodies were used for validation. The black arrow denotes the upper band as Akt3-GFP. The white arrow denotes Akt3v-GFP mixed with the unspecific band. (F) The percentage of GFP positive DU-L cells quantified by flow cytometry. (G) The percentage of c-casp3 positive DU-L populations in GFP+ cells upon CPT + TRAIL treatment for 3 h by flow cytometry. (H) The percentage of GFP positive DU-H cells quantified by flow cytometry. (I) The percentage of the c-casp3 positive DU-H population in GFP+ cells upon CPT + TRAIL treatment for 4 h by flow cytometry. Data shown are the means ± SD. Student’s t-test was used for comparative analyses. *P < 0.05; **P < 0.01; ***P < 0.001. One representative experiment (of at least 3 independent repeats) is presented in the immunoblotting panels.
Fig 5: TR4 modulates seminoma proliferation and metastasis through altering the AKT3 expression. (A) analysis of mRNA co-expression: TR4 and AKT3 by cBio Cancer Genomics Portal (N = 65). (B) The AKT3 mRNA and protein level in Tcam-2-TR4 and control cells are detected by quantitative RT-PCR and western blot assays. (C) AKT3 immunofluorescence assay results. Tcam-2-TR4 and control cells are analyzed. (D) Analysis of AKT3 mRNA expression with different clinical tumor stage. ns, no significance
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