Journal: Frontiers in oncology

Article Title: Circulating miR-320a Acts as a Tumor Suppressor and Prognostic Factor in Non-small Cell Lung Cancer.

doi: 10.3389/fonc.2021.645475

Figure Lengend Snippet: FIGURE 6 | MiR-320a regulates the PI3K/AKT/mTOR pathway through its direct target, AKT3. (A) MiR-320a in silico target prediction shows 124 common targets (inside black circle) common to databases starBase (peach), miRDB (blue), TargetScan version 7.2 (green), and DIANA micro-T (pink). (B) MiR-320a binding site with AKT3 as predicted by DIANA micro-T, an online computational tool. (C) Dual-luciferase reporter assays show miR-320a regulating AKT3 through direct binding. The data are presented as mean ± SEM from three experiments where *p > 0.05, **p < 0.05 significant differences from negative control (NC) calculated using paired t-test. (D) Immunoblot assays (a representative blot is shown) were used to determine normalized protein levels of PI3K, AKT3, Phospho-AKT3 (pThr305), mTOR, and Phospho-mTOR (pSer2448) proteins in untransfected A549 cells, A549 cells transfected with the vehicle control, a miR-320a inhibitor (100 nM), or mimic (100 nM). All experiments were carried out four independent times. The data are presented as mean ± SEM where *p > 0.05, **p < 0.05 significant differences from vehicle control calculated using paired t-test. (E) Immunohistochemistry images represent tumor cells positive (brown-stained) for PI3K, AKT3, pAKT3, mTOR, and p-mTOR proteins in the cytoplasm of NSCLC patient tissue samples. Scale bar 100 µm and n = 3 NSCLC tissues.

Article Snippet: Approximately 30 μg of protein extract was used to evaluate the target protein levels using primary antibodies of anti-AKT3 (Cat. No.: 14982), anti-pAKT (pThr308) (Cat. No.: 13038), AKT3 phosphorylated at Thr305, anti-PI3K p110α (Cat. No.: 4249), anti-mTOR (Cat. No.: 2983), anti-phospho-mTOR (pSer2448) (Cat. No.: 5536), anti-Caspase 3 (Cat. No.:9662), anti-p27 (Cat. No.: 3686), antiBeta-catenin (Cat. No.:8480), anti-MMP9 (Cat. No.: 13667), anti-E-cadherin (Cat. No.: 3195) purchased from Cell Signaling Technologies, Inc., Danvers, Massachusetts, USA, while antiCyclin D1 (Cat. No.: C7464), anti-Bcl-2 (Cat. No.: SAB4500003) were purchased from Sigma-Aldrich, Inc., St. Louis, MO, USA.

Techniques: In Silico, Binding Assay, Luciferase, Negative Control, Western Blot, Transfection, Control, Immunohistochemistry, Staining

Journal: Frontiers in oncology

Article Title: Circulating miR-320a Acts as a Tumor Suppressor and Prognostic Factor in Non-small Cell Lung Cancer.

doi: 10.3389/fonc.2021.645475

Figure Lengend Snippet: FIGURE 7 | Illustration of the potential mechanistic roles of miR-320a in NSCLC pathogenesis. Circulating miR-320a binding to the 3′UTR of AKT3 results in an increase or decrease in its downstream molecules represented by up and down arrows, respectively, thereby, influencing the AKT3-associated molecules to impact hallmarks of cancer in NSCLC tumorigenesis.

Article Snippet: Approximately 30 μg of protein extract was used to evaluate the target protein levels using primary antibodies of anti-AKT3 (Cat. No.: 14982), anti-pAKT (pThr308) (Cat. No.: 13038), AKT3 phosphorylated at Thr305, anti-PI3K p110α (Cat. No.: 4249), anti-mTOR (Cat. No.: 2983), anti-phospho-mTOR (pSer2448) (Cat. No.: 5536), anti-Caspase 3 (Cat. No.:9662), anti-p27 (Cat. No.: 3686), antiBeta-catenin (Cat. No.:8480), anti-MMP9 (Cat. No.: 13667), anti-E-cadherin (Cat. No.: 3195) purchased from Cell Signaling Technologies, Inc., Danvers, Massachusetts, USA, while antiCyclin D1 (Cat. No.: C7464), anti-Bcl-2 (Cat. No.: SAB4500003) were purchased from Sigma-Aldrich, Inc., St. Louis, MO, USA.

Techniques: Binding Assay

Journal: Journal of Virology

Article Title: Specific Akt Family Members Impair Stress-Mediated Transactivation of Viral Promoters and Enhance Neuronal Differentiation: Important Functions for Maintaining Latency

doi: 10.1128/jvi.00901-20

Figure Lengend Snippet: Figure 4. Akt3 efficiently promotes neurite formation in Neuro-2A cells. 730

Article Snippet: A plasmid that expresses Akt3 was a gift from William Sellers (1236 320 pcDNA3 Myr HA Akt3; plasmid 9017; Addgene).

Techniques:

Journal: BioMed Research International

Article Title: Knockdown of AKT3 ( PKB γ ) and PI3KCA Suppresses Cell Viability and Proliferation and Induces the Apoptosis of Glioblastoma Multiforme T98G Cells

doi: 10.1155/2014/768181

Figure Lengend Snippet: Effect of siRNA on T98G cells. ((a), (b)) Comparison of mRNA copy numbers for AKT3 (a) and PI3KCA (b) between transfected and untransfected T98G cells ( AKT3 : P < 0.01, t = 4.178, β = 0.981; PI3KCA : P < 0.01, t = 4.98, β = 0.987); ((c), (d)) dot-plots for comparison of the percentage of AKT3 protein-positive cells after AKT3 and PI3KCA knockdown and without using AKT3 siRNA; analysis was performed using flow cytometry; ((e), (f)) comparison of the percentage of AKT3 or PI3KCA protein-positive cells between the groups that were analyzed ( P < 0.05).

Article Snippet: The cells were then washed twice in PBS with 1% BSA, permeabilized with 0.1% saponin/1% BSA/PBS with 10% goat serum for 45 min, and incubated with 10 μ g/mL of an anti-AKT3 monoclonal antibody (isotype IgG 2A , R&D Systems, Inc.) in 1% BSA/PBS overnight at 4°C.

Techniques: Comparison, Transfection, Knockdown, Flow Cytometry

Journal: BioMed Research International

Article Title: Knockdown of AKT3 ( PKB γ ) and PI3KCA Suppresses Cell Viability and Proliferation and Induces the Apoptosis of Glioblastoma Multiforme T98G Cells

doi: 10.1155/2014/768181

Figure Lengend Snippet: Reduction in the AKT3 and PI3KCA protein expression in T98G cells after AKT3 and PI3KCA knockdown compared to untransfected cells. All of the figures are representative of three independent experiments. Data are expressed as means ± SEM ( P < 0.05).

Article Snippet: The cells were then washed twice in PBS with 1% BSA, permeabilized with 0.1% saponin/1% BSA/PBS with 10% goat serum for 45 min, and incubated with 10 μ g/mL of an anti-AKT3 monoclonal antibody (isotype IgG 2A , R&D Systems, Inc.) in 1% BSA/PBS overnight at 4°C.

Techniques: Expressing, Knockdown

Journal: BioMed Research International

Article Title: Knockdown of AKT3 ( PKB γ ) and PI3KCA Suppresses Cell Viability and Proliferation and Induces the Apoptosis of Glioblastoma Multiforme T98G Cells

doi: 10.1155/2014/768181

Figure Lengend Snippet: Cytotoxic effect of AKT3 and PI3KCA siRNAs on T98G cell viability as determined by trypan blue exclusion and the WST-1 assay ( P < 0.05).

Article Snippet: The cells were then washed twice in PBS with 1% BSA, permeabilized with 0.1% saponin/1% BSA/PBS with 10% goat serum for 45 min, and incubated with 10 μ g/mL of an anti-AKT3 monoclonal antibody (isotype IgG 2A , R&D Systems, Inc.) in 1% BSA/PBS overnight at 4°C.

Techniques: WST-1 Assay

Journal: BioMed Research International

Article Title: Knockdown of AKT3 ( PKB γ ) and PI3KCA Suppresses Cell Viability and Proliferation and Induces the Apoptosis of Glioblastoma Multiforme T98G Cells

doi: 10.1155/2014/768181

Figure Lengend Snippet: (a) DNA content frequency histograms representing cells from untransfected cultures and from cultures that had been transfected with AKT3 , PI3KCA , and AKT3 + PI3KCA siRNAs. (b) The cell cycle distribution of the T98G cell culture that had been transfected with AKT3 , PI3KCA , and AKT3 + PI3KCA siRNAs, respectively, compared to the untransfected control cells. Cells were fixed in 70% ethanol and stained with PI. Fluorescence of the PI-stained cells was measured using a FACS AriaII cytometer (Becton Dickinson).

Article Snippet: The cells were then washed twice in PBS with 1% BSA, permeabilized with 0.1% saponin/1% BSA/PBS with 10% goat serum for 45 min, and incubated with 10 μ g/mL of an anti-AKT3 monoclonal antibody (isotype IgG 2A , R&D Systems, Inc.) in 1% BSA/PBS overnight at 4°C.

Techniques: Transfection, Cell Culture, Control, Staining, Fluorescence, Cytometry

Journal: BioMed Research International

Article Title: Knockdown of AKT3 ( PKB γ ) and PI3KCA Suppresses Cell Viability and Proliferation and Induces the Apoptosis of Glioblastoma Multiforme T98G Cells

doi: 10.1155/2014/768181

Figure Lengend Snippet: Comparison of the DNA index (the ratio of transfected cell DNA fluorescence/untransfected G1-G0 cell DNA fluorescence) between cells that had been transfected with AKT3 , PI3KCA , and AKT3 + PI3KCA siRNAs and untransfected cells ( P < 0.05). A DNA index of one corresponds to diploidy (2C).

Article Snippet: The cells were then washed twice in PBS with 1% BSA, permeabilized with 0.1% saponin/1% BSA/PBS with 10% goat serum for 45 min, and incubated with 10 μ g/mL of an anti-AKT3 monoclonal antibody (isotype IgG 2A , R&D Systems, Inc.) in 1% BSA/PBS overnight at 4°C.

Techniques: Comparison, Transfection, Fluorescence

Journal: BioMed Research International

Article Title: Knockdown of AKT3 ( PKB γ ) and PI3KCA Suppresses Cell Viability and Proliferation and Induces the Apoptosis of Glioblastoma Multiforme T98G Cells

doi: 10.1155/2014/768181

Figure Lengend Snippet: Comparison of the proliferation index between cells that had been transfected with AKT3 , PI3KCA, or AKT3 + PI3KCA siRNAs and untransfected cells ( P < 0.05).

Article Snippet: The cells were then washed twice in PBS with 1% BSA, permeabilized with 0.1% saponin/1% BSA/PBS with 10% goat serum for 45 min, and incubated with 10 μ g/mL of an anti-AKT3 monoclonal antibody (isotype IgG 2A , R&D Systems, Inc.) in 1% BSA/PBS overnight at 4°C.

Techniques: Comparison, Transfection

Journal: BioMed Research International

Article Title: Knockdown of AKT3 ( PKB γ ) and PI3KCA Suppresses Cell Viability and Proliferation and Induces the Apoptosis of Glioblastoma Multiforme T98G Cells

doi: 10.1155/2014/768181

Figure Lengend Snippet: BAX/BCL-2 ratio in T98G cells after AKT3 , PI3KCA , and AKT3 + PI3KCA gene silencing using specific siRNAs ( P < 0.05).

Article Snippet: The cells were then washed twice in PBS with 1% BSA, permeabilized with 0.1% saponin/1% BSA/PBS with 10% goat serum for 45 min, and incubated with 10 μ g/mL of an anti-AKT3 monoclonal antibody (isotype IgG 2A , R&D Systems, Inc.) in 1% BSA/PBS overnight at 4°C.

Techniques:

Journal: BioMed Research International

Article Title: Knockdown of AKT3 ( PKB γ ) and PI3KCA Suppresses Cell Viability and Proliferation and Induces the Apoptosis of Glioblastoma Multiforme T98G Cells

doi: 10.1155/2014/768181

Figure Lengend Snippet: The influence of AKT3 and PI3KCA gene knockdown on the induction of apoptosis. The datasheets show the apoptosis and necrosis rates in the groups that were analyzed (a) and the value of the apoptotic index (b). The data are expressed as the means of three separate experiments ( P < 0.05).

Article Snippet: The cells were then washed twice in PBS with 1% BSA, permeabilized with 0.1% saponin/1% BSA/PBS with 10% goat serum for 45 min, and incubated with 10 μ g/mL of an anti-AKT3 monoclonal antibody (isotype IgG 2A , R&D Systems, Inc.) in 1% BSA/PBS overnight at 4°C.

Techniques: Knockdown