Journal: Nature Communications

Article Title: Recurrent hotspot mutations in HRAS Q61 and PI3K-AKT pathway genes as drivers of breast adenomyoepitheliomas

doi: 10.1038/s41467-018-04128-5

Figure Lengend Snippet: Impact of AKT and MEK inhibition on PI3K-AKT and MAPK signaling pathways and proliferation in non-malignant breast epithelial cells expressing mutant HRAS Q61R . a Representative western blot analysis of p-ERK1/2 (T202/Y204), p-p90 RSK (S380), p-AKT (S473), p-AKT (T308), p-PRAS40 (T246), p-FOXO1/3a/4, p-GSK3β (S9), p-mTOR (S2448), p-p70 S6K (T389), and p-S6 (S240/244) protein in MCF-10A P and MCF-10A H1047R cells stably expressing empty vector (EV) or mutant HRAS Q61R treated with 2 µM AKT inhibitor (AKTi, MK2206) at different time points. β-actin was used as a protein loading control. Experiments were repeated at least twice with similar results . b Cell proliferation assay of MCF-10A P and MCF-10A H1047R cells stably expressing EV or mutant HRAS Q61R . **** P < 0.0001; two-tailed unpaired t -test. c Inhibition effects of cells treated with DMSO (black), 1 µM AKTi (blue), 10 nM MEK inhibitor (MEKi, GSK212, green), and combination of 1 µM AKTi and 10 nM MEKi (red) for 3, 5, and 7 days. In b and c , cells were cultured in growth factor- and serum-free media. Data are representative of three independent experiments. Error bars, s.d. of mean ( n = 3). d Representative micrographs of MCF-10A P and MCF-10A H1047R cells stably expressing EV or mutant HRAS Q61R cultured after 3 days and treated with DMSO, 1 µM AKTi, 10 nM MEKi, or combination of 1 µM AKT and 10 nM MEK inhibitors for 6 and 9 days are shown (scale bars, 400 µm). DMSO or inhibitors were added after seeding the cells in three-dimensional basement membrane for 3 days; fresh media with DMSO or inhibitors was replenished every 3 days. Triplicate experiments were repeated at least twice with similar results

Article Snippet: The AKT inhibitor MK2206 (MedChem Express HY-10358) and MEK inhibitor GSK212 (Selleck Chemicals s2658) were employed for pharmacological inhibition in cell lines analyzed in in vitro experiments time points and concentrations indicated.

Techniques: Inhibition, Protein-Protein interactions, Expressing, Mutagenesis, Western Blot, Stable Transfection, Plasmid Preparation, Control, Proliferation Assay, Two Tailed Test, Cell Culture, Membrane

Journal: International Journal of Biological Sciences

Article Title: Accelerated Bone Regeneration by Astragaloside IV through Stimulating the Coupling of Osteogenesis and Angiogenesis

doi: 10.7150/ijbs.57681

Figure Lengend Snippet: AS-IV activates the AKT/GSK-3β/β-catenin pathway in BMSCs. (A) Western blot of AKT, p-AKT, GSK-3β, p-GSK-3β, β-catenin, and p-β-catenin in BMSCs treated with OIM, OIM+AS-IV, and OIM+AS-IV+MK2206. (B) Quantitative analysis of the phosphorylated levels of AKT, GSK-3β, and β-catenin. (C) Representative immunocytochemistry images showing the expression and nuclear translocation of β-catenin in BMSCs treated with OIM, OIM+AS-IV, and OIM+AS-IV+MK2206. Scale bar: 100 µm. (D) Quantitative analysis of the expression and nuclear translocation of β-catenin. ** P < 0.01. MFI, mean fluorescence intensity. The data were confirmed by one-way analysis of variance (ANOVA) followed by Tukey's post hoc test and are presented as the means ± SD. NS P > 0.05; ** P < 0.01.

Article Snippet: Additionally, MK2206 (100 nM; MedChemExpress, USA) was administered to verify the molecular mechanism by which AS-IV regulates cell viability of BMSC.

Techniques: Western Blot, Immunocytochemistry, Expressing, Translocation Assay, Fluorescence

Journal: International Journal of Biological Sciences

Article Title: Accelerated Bone Regeneration by Astragaloside IV through Stimulating the Coupling of Osteogenesis and Angiogenesis

doi: 10.7150/ijbs.57681

Figure Lengend Snippet: MK2206 inhibits the effects of AS-IV on osteogenic differentiation, cell viability, and angiogenic gene expression of BMSCs. (A-D) Osteogenesis of BMSCs treated with OIM, OIM+AS-IV, and OIM+AS-IV+MK2206 was determined with ALP staining (A), ALP activity assays (B) and alizarin red staining (C). Calcium deposition was assessed by measuring the optical density (D). Scale bar: 200 µm. (E) CCK-8 analysis of BMSC proliferation in different treatment groups. (F, G) Expression of osteogenic-specific genes (F) and angiogenic-specific genes (G) of BMSCs in different treatment groups were assessed using qRT-PCR. The data were confirmed by one-way analysis of variance (ANOVA) followed by Tukey's post hoc test and are presented as the means ± SD. ** P < 0.01; # P < 0.05 vs control group; ## P < 0.01 vs control group; ΔΔ P < 0.01 vs AS-IV group.

Article Snippet: Additionally, MK2206 (100 nM; MedChemExpress, USA) was administered to verify the molecular mechanism by which AS-IV regulates cell viability of BMSC.

Techniques: Gene Expression, Staining, Activity Assay, CCK-8 Assay, Expressing, Quantitative RT-PCR, Control

Journal: Nature Communications

Article Title: Cdk5 and GSK3β inhibit fast endophilin-mediated endocytosis

doi: 10.1038/s41467-021-22603-4

Figure Lengend Snippet: a Scoring criteria used in the kinase screen. Representative images of decreased, normal, and increased FEME in resting human RPE1 cells treated with 10 μM dobutamine, 10 μM DMSO, and 10 nM GDC-0941 (PI3Ki), respectively. Arrowheads point at FEME carriers. Decreased FEME was assigned for samples with >80% reduction in the number of cytoplasmic Endophilin-positive assemblies (EPAs), in at least 50% of the cells. Increased FEME was attributed to samples with >200% elevation in the number of EPAs, in at least 50% of the cells. The corresponding scoring marks were 0, 1, and 2, respectively. Scale bar, 5 μm. b Kinase screen using small compound inhibitors. RPE1 cells grown in complete medium were incubated for 10 min at 37 °C with the following inhibitors: DMSO, (vehicle); dobutamine, 10 μM (positive control); Dinaciclib (Cdk1/2/5/9i), 1 μM; CHIR-99041 (GSK3i1), 1 μM; BIO (GSK3i2), 1μM; Roscovitine (Cdk1/2/5i), 1 μM; PHA-793887 (Cdk2/5/7i), 100 nM; VX-745 (p38i), 10 μM; JNK-IN-8 (JNKi), 1 μM; staurosporine (broad kinases), 1 μM; GNE-7915 (LRRK2i), 1 μM; AZ191 (DYRK1Bi), 10 μM; GSK2334470 (PDKi), 10 μM; PF-4708671 (p70S6Ki), 10μM; AZ191 (DYRKi), 10 μM; AZD0530 (broad SRCi), 1 μM; TAK-632 (panRAFi), 10 μM; GW 5074 (CRAFi), 1 μM; PD0332991 (Cdk4/6i), 1 μM; MK2206 (AKTi), 1 μM; GDC-0879 (BRAFi), 1 μM; CX-4945 (CK2i), 1 μM; ZM 447439 (AurA/AurBi), 1 μM; RO-3306 (Cdk1i), 100 nM; BI 2536 (PLKi), 1 μM; PD0325901 (MEKi), 100 nM; Genistein (Y-kinases), 1 μM; Purvalanol A (Cdk1/2/4i), 100 nM; MLR 1023 (LYNi), 1 μM; P505-15 (SYKi), 100 nM; CDK1/2 inhibitor III (Cdk1/2i), 100 nM; KT 5720 (PKAi), 100 nM; BI-D1870 (p90RSKi), 100 nM; D4476 (CK1E), 1 μM; PF-4800567 (CK1Ei), 1 μM; SCH772984 (ERKi), 100 nM; STO609 (CaMKK1/2ii), 100 nM; P505-15 (SYKi), 1μM; PND-1186 (FAKi), 100 nM; Torin 1 (mTORC1/2i), 10 μM and GDC-0941 (PI3Ki), 100 nM (negative control). Histograms show the mean ± SEM from 12 well per condition, from three independent biological experiments. Statistical analysis was performed by one-way ANOVA. ns non significant; * P < 0.05, ** P < 0.01. c Number of FEME carriers (EPAs) upon titration of CHIR-99021, BIO, Roscovitine and Dinaciclib. Dobutamine and GDC-0941 were used as positive and negative controls, respectively. Plots show the mean ± SEM from three cells per condition and per timepoint, from three independent biological experiments. d β1-adrenergic receptor (β1AR) uptake into FEME carriers in RPE1 cells pre-treated with 5 μM CHIR-99021 (GSK3i) for 5 min, followed by 10 μM dobutamine for 4 min or not (resting). Scale bars, 5 μm. Histograms show the mean ± SEM of the number of FEME carriers (LHS: left hand side) and the number of FEME carriers positive for β1AR per 100 μm 2 (RHS: right hand side) ( n = 30 cells per condition, from biological triplicates). Arrowheads point at FEME carriers. Statistical analysis was performed by two-way ANOVA. ns non significant; * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: The following small compound inhibitors (amongst the best-reported inhibitors for each kinase , ) were used: AZ191 (called DYRKi in this study Cayman 17693), AZD0530 aka Sacratinib (called SRCi in this study, Cayman 11497), BI-D1870 (called p90RSKi in this study, Cayman 15264), BIO-6-bromoindirubin-3′-oxime, aka BIO (called GSK3i2 in this study, (Sigma B1686), BI 2536 (called PLKi in this study, Selleckchem S1109), CDK1/2 inhibitor III (called Cdk1/2i in this study, Merck 217714), CHIR-99041 (called GSK3i1 in this study, Cayman 13122), Ciliobrevin D (called Ciliobrevin in this study, Calbiochem 250401), CX-4945 (called CK2i in this study, Cayman 16779), Dinaciclib (called Cdk1/2/5/9i in this study, MedChemExpress Hy-10492), Dobutamine (Sigma D0676), D4476 (called CK1i in this study, BioVision 1770), GDC-0879 (called BRAFi in this study, Tocris 4453), GDC-0941 (called PI3Ki in this study, Symansis SYG0941), Genistein (called Y-kinases in this study, Calbiochem 245834), GNE-7915 (called LRRK2i in this study, MedChemExpress Hy-10328), GSK2334470 (called PDKi in this study, Cayman 18095), GW 5074 (called CRAFi in this study, Santa Crux sc-200639), Harmine hydrochloride (called DYRKi in this study, Santa Crux sc2595136), JNK-IN-8 (called JNKi in this study MedChemExpress Hy-13319), KT 5720 (called PKAi in this study Cayman 10011011), MK2206 (called AKTi in this study, LKT Laboratories M4000), MLR 1023 (called LYNa in this study, Tocris 4582), PD0325901 (called MEKi in this study, Tocris 4192), PD0332991 aka Palbociclib (called Cdk4/6i in this study, Sigma PZ0199), PF-4708671 (called p70S6Ki in this study, MedChemExpress Hy-15773), PF-4800567 (called CK1Ei in this study, Cayman 19171), PHA-793887 (called Cdk2/5/7i in this study, ApexBio A5459), PND-1186 (called FAKi in this study, MedChemExpress Hy-13917), Purvalanol A (called Cdk1/2/4i in this study, Santa Cruz sc-224244), P505-15 (called SYKi in this study, Adooq Bioscence A11952), Roscovitine (called Cdk1/2/5i in this study, Santa Cruz sc-24002), RO-3306 (called Cdk1i in this study, Cayman 15149), SCH772984 (called ERKi in this study, Sellekchem S7101), Staurosporine (called broad kinases in this study, Alomone Labs AM-2282), STO609 (called CaMKK1/2i in this study, Cayman 15325), TAK-632 (called panRAFi in this study, Selleckchem S7291), Torin 1 (called mTORC1i in this study, Tocris 4247), VX-745 (called p38i in this study, MedChemExpress Hy-10328) and ZM 447439 (called AurA/AurBi in this study, Cayman 13601).

Techniques: Incubation, Positive Control, Negative Control, Titration

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: 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: 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