Journal: iScience

Article Title: Global-run on sequencing identifies Gm11967 as an Akt-dependent long noncoding RNA involved in insulin sensitivity

doi: 10.1016/j.isci.2022.104410

Figure Lengend Snippet:

Article Snippet: MK2206 , Toronto Research Chemicals , Cat# M425025.

Techniques: Virus, Plasmid Preparation, Recombinant, Injection, Protease Inhibitor, One Step RT-PCR, SYBR Green Assay, Enzyme-linked Immunosorbent Assay, Bicinchoninic Acid Protein Assay, Software, Real-time Polymerase Chain Reaction

Journal: International journal of cancer

Article Title: Effects of Metformin on the virus/host cell crosstalk in human papillomavirus-positive cancer cells.

doi: 10.1002/ijc.33594

Figure Lengend Snippet: FIGURE 2 Effects of glucose, AKT inhibitors and PI3K inhibitors on Metformin-induced E6/E7 repression. (A) Immunoblot analyses of E6/E7 levels in HeLa and SiHa cells treated for 24 hours with the indicated doses of Metformin under varying glucose concentrations. Vinculin, representative loading control. (B) Immunoblot analyses comparing E6/E7, phospho-AKT (P-AKTS473) and total AKT expression levels in Metformin- and hypoxia-treated cervical cancer cells. HeLa and SiHa cells are cultivated for 24 hours under normoxia in the absence of Metformin (left panels), under 2.5 mM or 5 mM Metformin, respectively (central panels), or under hypoxic conditions (1% O2) (right panels). Additionally, high glucose (25 mM) or 10 μM of the AKT inhibitor AKTi VIII are added when indicated. Vinculin, representative loading control. (C) Analyses of the effects of a spectrum of AKT inhibitors (AKTi VIII, MK-2206, Ipatasertib, GSK-690693, Afuresertib) and PI3K inhibitors (LY294002, PX-886 and GDC-0941) on Metformin (central panel)- and hypoxia (right panel)-induced E6/E7 repression and on P-AKTS473, P-AKTT308 and total AKT levels. HeLa cells are cultivated for 24 hours under normoxia in the absence of Metformin (left panel), under 2.5 mM Metformin (central panel) or under hypoxic conditions (1% O2) (right panel). Additionally, high glucose (25 mM), AKT inhibitors or PI3K inhibitors are added when indicated (for individual concentrations of the inhibitors please refer to Section 2). Vinculin, β-Actin, representative loading controls

Article Snippet: PI3K and AKT inhibitors were employed at the following end concentrations: 10 μM AKTi VIII (Sigma Aldrich), 10 μM MK-2206 (Adipogen, Liestal, Switzerland), 10 μM Ipatasertib (MedChemExpress, Monmouth Junction, NJ), 10 μM GSK690693 (MedChemExpress), 5 μM Afuresertib (BioVision, Milpitas, CA), 20 μM LY294002 (Cayman Chemical, Ann Arbor, MI), 5 μM PX-886 (LKT Laboratories, St Paul, MN) and 5 μM GDC-0941 (Selleckchem, Houston, TX).

Techniques: Western Blot, Control, Expressing

Journal: Advanced Healthcare Materials

Article Title: 3D Bioprinted Coaxial Testis Model Using Human Induced Pluripotent Stem Cells:A Step Toward Bicompartmental Cytoarchitecture and Functionalization

doi: 10.1002/adhm.202402606

Figure Lengend Snippet: Immunostaining for SSC markers following the replacement of the neonatal mouse SSC growth factor EGF with Akt‐inhibitor MK2206 in hiPSC‐SSC cultures.

Article Snippet: SSCs were expanded on a sulfated dextran‐4‐armed polyethylene glycol (starPEG) hydrogel matrix functionalized with vitronectin and fibronectin type II domain (FN2) peptide motifs (denovoMATRIX, GmbH) in Human Plasma‐Like Medium (HPLM), 15% Cell Therapy Systems (CTS) KnockOut Serum Replacement (SR) XenoFree supplement (Gibco, 12 618 012), 1 μM vitamin C (Millipore Sigma, A4403), 1 μM vitamin E (Millipore Sigma, T1157), 10 μg mL −1 biotin (Millipore Sigma, B4639), 60 ng mL −1 progesterone (Millipore Sigma, P8783), 60 μM putrescine (Millipore Sigma, P5780), 30 μg mL −1 pyruvate (Millipore Sigma, S8636), 30 ng mL −1 β‐estradiol (Sigma, E2758), 1X Minimum Essential Medium (MEM) Vitamin Solution (Gibco, 11 120 052), 1X GlutaMAX Supplement (Gibco, 35 050 061), 1 μL mL −1 DL‐lactate (Millipore Sigma, L4263), 50 μM β‐mercaptoethanol (Gibco, 31350‐010), 15 ng mL −1 Glial Cell‐Derived Neurotrophic Factor (GDNF, Peprotech, AF‐450‐010), 10 ng mL −1 heat stable FGF2, and either 10 ng mL −1 EGF or 100 nM Akt pathway inhibitor MK2206 (Biogems, 1 031 320).

Techniques: Immunostaining

Journal: bioRxiv

Article Title: The Hexosamine Biosynthetic Pathway alters the cytoskeleton to modulate cell proliferation and migration in metastatic prostate cancer

doi: 10.1101/2024.10.14.618283

Figure Lengend Snippet: (A-C) Control and GNPNAT1 KO 22Rv1 cell lysate samples were Western blot-analyzed for the levels of AKT and its phosphorylated form p-AKT (A) as well as AKT downstream signaling pathways including components of the mTOR pathway (B), and the PKC pathway (C). (D-E) Microscopic images (D) and cell proliferation assay (E) after treating control and GNPNAT1 KO with the AKT inhibitor, MK2206 after 48 h. Scale bars, 200 µm. F) Cell proliferation assay after treatment with UDP-GlcNAc after 48 h. (G) Cell proliferation assay after pre-treatment with 10 µM MK2206 followed by co-treatment with 10 µM MK2206 and 30 µM UDP-GlcNAc or UDP-GlcNAc alone. (H) Schematic diagram of the possible mechanism for increased cell proliferation in GNPNAT1 KO cells, via the alteration of AKT and its downstream signaling pathways.

Article Snippet: MK2206 was purchased from AdooQ Bioscience (Irvine, CA) and UDP-GlcNAc from Millipore sigma (Burlington, MA).

Techniques: Control, Western Blot, Proliferation Assay

Journal: Nature Communications

Article Title: EGFR-mediated activation of adipose tissue macrophages promotes obesity and insulin resistance

doi: 10.1038/s41467-022-32348-3

Figure Lengend Snippet: Hyperinsulinemic-euglycemic clamps were performed on 5 h fasted WT and MΦ EGFR −/− mice after 12 weeks on the HFD. A MΦ EGFR −/− mice had lower plasma insulin levels at baseline and during clamp periods. N = 5 and 7. B MΦ EGFR −/− mice had less severe insulin resistance, as more glucose infusion was needed to maintain a constant blood glucose. N = 5 and 7. C , D . MΦ EGFR −/− mice had increased rates of glucose disappearance (Rd) ( N = 4 and 7) C and decreased endogenous glucose production (EGP) ( N = 4 and 7) D . E MΦ EGFR −/− mice had increased glucose uptake, a marker of insulin resistance in VAT and SA. N = 5 and 7. F Representative images showed more insulin-stimulated p-Akt in EF in MΦ EGFR −/− mice. Scale bar = 100 μm. G Immunoblotting determined higher insulin-stimulated p-Akt in EF in MΦ EGFR −/− mice. N = 3 and 4). Data are means ± SEM, * P < 0.05, ** P < 0.01, *** P < 0.001, analyzed using 2-way ANOVA followed by Bonferroni’s post hoc test for A , and C – E ; 2-way ANOVA followed by Tukey’s post hoc test for B ; and 2 tailed Student’s t test for G .

Article Snippet: Cells translocated to the lower chamber in response to exposure in the lower chamber of vehicle or 100 ng/ml CX3CL1 (472-FF; R&D) in the presence or absence of either an AKT inhibitor (MK-2206; 10 μM, Cayman), PI3K inhibitor (BEZ235; 10 μM, Cayman), EGFR inhibitor (erlotinib, 10 μM, LC laboratories) or NF-κB inhibitor (JSH23, 30 μM, Sigma-Aldrich) for 3 h. Cells in the upper chamber were removed with a cotton swab, and the filters were fixed with 70% ethanol and stained with 2% crystal violet.

Techniques: Clinical Proteomics, Marker, Western Blot

Journal: American Journal of Cancer Research

Article Title: High expression of peroxisomal D-bifunctional protein in cytosol regulates apoptosis and energy metabolism of hepatocellular carcinoma cells via PI3K/AKT pathway

doi:

Figure Lengend Snippet: Cytosolic DBP promoted HepG2 cell proliferation via PI3K/AKT/FOXO3a/Bim pathway. A, B. Representative WB images of DBP, p-AKT, AKT, p-FOXO3a, FOXO3a, p-JNK, JNK, and Bim. β-actin served as a loading control. The quantitation of WB data was presented. C. Representative WB images of FOXO3a in the nuclear fraction. LAMN and GAPDH were nuclear and cytosolic markers, respectively. The quantitation of WB data was presented. D. The IF staining of FOXO3a (red), DAPI (blue), and their merge (purple). Quantitation of PCC data was presented. Scale bars = 10 μm. E. qRT-PCR to detect the expression of Bim. F. Apoptosis was assessed by flow cytometry with propidium iodide (PI)/Annexin V staining and the corresponding apoptosis ratio (%). Q2 region indicated late apoptotic cells with necrosis cells and mechanically damaged cells. Q4 indicated early apoptotic cells. G. Colony formation assay. H. CCK-8 assay. HepG2 cells were infected with control adenovirus (Empty), DBP-SKL, or DBP-deSKL. HepG2 cells were also treated with non-specific siRNA (siNC), DBP specific siRNA (siDBP), or AKT specific siRNA (siAKT). MK: MK2206, AKT inhibitor. LY: LY294002, PI3K inhibitor. Data were presented as mean ± SD. *P<0.05, **P<0.01 and ***P<0.001 vs. Empty group; #P<0.05, ##P<0.01 and ###P<0.001 vs. DBP-SKL group; •P<0.05, ••P<0.01 and •••P<0.001 vs. DBP-deSKL group.

Article Snippet: Phosphoinositide 3-kinase (PI3K) inhibitor LY294002 (GC15485; 20 mmol/L) and AKT inhibitor MK2206 (GC16304; 10 mmol/L) were purchased from GlpBio, New York, USA.

Techniques: Control, Quantitation Assay, Staining, Quantitative RT-PCR, Expressing, Flow Cytometry, Colony Assay, CCK-8 Assay, Infection

Journal: American Journal of Cancer Research

Article Title: High expression of peroxisomal D-bifunctional protein in cytosol regulates apoptosis and energy metabolism of hepatocellular carcinoma cells via PI3K/AKT pathway

doi:

Figure Lengend Snippet: Mitochondrial localization of DBP was p-AKT-dependent. (A-C) The IF staining of DBP (red) in MK2206-treated DBP-SKL- and DBP-deSKL-expressing HepG2 cells. Cells were also co-stained with peroxisomal marker PMP70 (green) (A), mitochondrial marker COXIV (green) (B), and GAPDH (green) (C). Scale bars = 10 μm. PCC data were quantified. (D) WB of p-DBP in MK2206-treated mitochondria of DBP-SKL- or DBP-deSKL-overexpressing HepG2 cells. (E) CO-IP of DBP and p-Akt in DBP-SKL-expressing cells. (F) The IF staining of DBP (red) with p-AKT (green) in MK2206-treated DBP-SKL-expressing HepG2 cells. PCC data were quantified. Scale bar = 10 μm. (G) WB of p-DBP in tumor tissues and the adjacent liver tissues. (H) WB of p-DBP in DBP-SKL-overexpressing cells. HepG2 cells were infected with control adenovirus (Empty), DBP-SKL, or DBP-deSKL. MK: MK2206, AKT inhibitor. Data were presented as mean ± SD. ***P<0.001 vs. Empty group; ###P<0.001 vs. DBP-SKL group; •••P<0.001 vs. DBP-deSKL group.

Article Snippet: Phosphoinositide 3-kinase (PI3K) inhibitor LY294002 (GC15485; 20 mmol/L) and AKT inhibitor MK2206 (GC16304; 10 mmol/L) were purchased from GlpBio, New York, USA.

Techniques: Staining, Expressing, Marker, Co-Immunoprecipitation Assay, Infection, Control

Journal: American Journal of Cancer Research

Article Title: High expression of peroxisomal D-bifunctional protein in cytosol regulates apoptosis and energy metabolism of hepatocellular carcinoma cells via PI3K/AKT pathway

doi:

Figure Lengend Snippet: Cytosolic DBP regulated the production of glycogen and ATP in HepG2 cells via PI3K/AKT/GSK3β signaling pathway. A, B. WB of p-GSK3β and GSK3β. β-actin: loading control. Data were quantified. C. Glycogen levels in the indicated cells. D. Glucose uptake as indicated by 2-NBDG fluorescence intensity in the treated cells. Scale bar (left) = 100 μm, scale bar (right) = 30 μm. E. Glycogen staining by PAS in tumors and the adjacent liver tissues of HCC patients (n = 4). PAS-positive staining (magenta) was marked by arrows. Scale bars = 100 μm. F. WB of p-GSK3β in the mitochondrial fraction of cells. COXIV and β-actin: mitochondrial and cytosolic markers, respectively. Data were quantified. G. The IF staining of p-GSK3β (green), mitochondrial COXIV (red), and their merge (yellow). PCC data were quantified. Scale bars = 10 μm. H. The enzymatic activity of mitochondrial respiratory chain complex III in the treated cells. I. ATP levels in the treated cells. HepG2 cells were infected with control adenovirus (Empty), DBP-SKL, or DBP-deSKL. HepG2 cells were also treated with non-specific siRNA (siNC), DBP specific siRNA (siDBP), AKT specific siRNA (siAKT). MK: MK2206, AKT inhibitor. Data were presented as mean ± SD. *P<0.05, **P<0.01 and ***P<0.001 vs. Empty group; #P<0.05, ##P<0.01 vs. DBP-SKL group; •P<0.05, ••P<0.01 and •••P<0.001 vs. DBP-deSKL group.

Article Snippet: Phosphoinositide 3-kinase (PI3K) inhibitor LY294002 (GC15485; 20 mmol/L) and AKT inhibitor MK2206 (GC16304; 10 mmol/L) were purchased from GlpBio, New York, USA.

Techniques: Control, Fluorescence, Staining, Activity Assay, Infection