Journal: The Journal of Biological Chemistry

Article Title: Selective export of autotaxin from the endoplasmic reticulum

doi: 10.1074/jbc.M116.774356

Figure Lengend Snippet: Effects of AKT inhibitor treatment and AKT knockdown on ATX secretion. A, HeLa cells were co-transfected with pcDNA3-ATX-Myc and pEGFP-N1-CB5 and then treated with or without AKT inhibitor MK-2206 (5 μm) for 24 h. ATX-Myc was visualized by confocal microscopy with anti-Myc (9E10) monoclonal antibody (red). Endoplasmic reticulum was labeled by EGFP-CB5 (green). Nuclei were counterstained with DAPI (blue). B, HeLa cells were transfected with pcDNA3-ATX-Myc and then treated with or without AKT inhibitor MK-2206 (5 μm) for 24 h. The concentrated (∼30-fold) serum-free conditional culture medium of the control cells and the lysate of AKT inhibitor-treated cells were treated with Endo H and then analyzed by Western blotting with anti-Myc antibody. C, HeLa cells with exogenous expression of ATX-Myc were treated with MK-2206 (5 μm) for 24 h or treated with siRNA against AKT for 48 h. Then ATX-Myc protein levels in cell lysates (L) and culture medium (M) were detected by immunoblotting with anti-Myc antibody. p23, AKT, and phosphorylated AKT (p-AKT) levels in cell lysates were detected by immunoblotting. D, HeLa cells with exogenous expression of ATX-Myc were treated with MK-2206 (5 μm) for 24 h or treated with siRNA against AKT for 48 h. ATX-Myc and p23 mRNA levels were detected by quantitative reverse transcription PCR (qRT-PCR). E, HeLa cells with exogenous expression of ATX-Myc and FLAG-p23 were treated with or without AKT inhibitor MK-2206 (5 μm) for 24 h. ATX-Myc protein levels in cell lysates and culture medium were detected by immunoblotting with anti-Myc antibody. FLAG-p23 levels in cell lysates were detected by immunoblotting with anti-FLAG antibody. F, HeLa cells were treated with MK-2206 (5 μm) for 24 h or treated with siRNA against AKT for 48 h. Then, NFAT1 levels in nuclear fraction (Nuc), cytoplasmic fraction (Cyto), and total cell lysates (Total) were detected by Western blotting. Lamin A and α-tubulin were used as the nuclear and cytoplasm markers, as indicated. G and H, HeLa cells with exogenous expression of ATX-Myc were transfected with nonspecific control siRNA (NC) or siRNA against AKT. Thirty two hours after transfection, cells were treated with or without GSK3β inhibitor SB415286 (25 μm) for 16 h. Then, ATX-Myc protein levels in cell lysates and culture medium, and p23 levels in cell lysates were detected by immunoblotting (G). p23 mRNA levels were detected by qRT-PCR (H). The p value was derived from analysis of variance. **, p < 0.005. Data are representative of three independent experiments.

Article Snippet: AKT inhibitor MK-2206 (S1078) and GSK3β inhibitor SB415286 were purchased from Selleck Chemicals (Houston, TX).

Techniques: Knockdown, Transfection, Confocal Microscopy, Labeling, Control, Western Blot, Expressing, Reverse Transcription, Quantitative RT-PCR, Derivative Assay

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: 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: Oncotarget

Article Title: Activation of RAS family members confers resistance to ROS1 targeting drugs

doi:

Figure Lengend Snippet: A: DNA sequence analysis of HCC78 parental cells (WT) and cells resistant to the ROS1 inhibitor JNJ-ROS1i-A (ROS1i-A-RES 4). KRAS codon 12 of WT (upper panel) and ROS1i-A-RES 4 cells (lower panel) is indicated. B: Cell growth of HCC78 cells transduced with either an empty vector (MOCK) or constructs for expression of KRAS G12V (left panel) or KRAS G12C (right panel). The cells were untreated (NT) or treated with 4μM JNJ-ROS1i-A (6 days of treatment). *: significantly different from control (* p< 0.05; ** p< 0.01). C: Cell growth of ROS1i-A-RES 4 cells transfected with the indicated siRNAs and grown in the presence of JNJ-ROS1i-A 4μM. ROS1 and NRAS siRNAs were used as additional controls. D: Growth of parental (WT) or resistant (ROS1i-A-RES 4) HCC78 cells, treated with the indicated concentrations of JNJ-ROS1i-A, crizotinib or foretinib; ns: not significant; **: significantly different from control (p< 0.01). E: Growth of HCC78 WT cells transduced with empty vector (MOCK) or a KRAS G12C expression construct, treated with either 4 μM JNJ-ROS1i-A, 1.4 μM crizotinib or 50nM foretinib. Cell growth was measured after 6 days of treatment; ns: not significant; **: significantly different from control (p< 0.01). F: Growth of HCC78 cells (WT or ROS1i-A-RES 4), treated for 6 days with the MAPK inhibitor U0126 (10 μM), the AKT inhibitor MK2206 (3 μM), alone or in combination. *: significantly different from control (* p< 0.05; ** p< 0.01).

Article Snippet: MAPK inhibitor (U0126) was purchased from Promega and the AKT inhibitor (Mk2206) from Merck (Whitehouse Station, NJ, USA).

Techniques: Sequencing, Transduction, Plasmid Preparation, Construct, Expressing, Transfection

Journal: Oncotarget

Article Title: Activation of RAS family members confers resistance to ROS1 targeting drugs

doi:

Figure Lengend Snippet: A: ROS1 mRNA levels in HCC78 cells transduced with empty vector (MOCK), KRAS G12C, KRAS or NRAS Q61K expression constructs. B: ROS1 protein (left panel) and mRNA (right panel) levels in HCC78 WT or ROS1 inhibitor resistant cells (ROS1i-A-RES 4 and CRIZ-RES 4), treated for 3 days with the MAPK inhibitor UO126. The left panel shows a WB of the treated cells, probed with the indicated antibodies. Vinculin was used as a loading control. The right panel shows ROS1 mRNA expression evaluated by qRT-PCT. ns: not significant; **: significantly different from control (p< 0.01). C: ROS1 protein (left panel) and mRNA (right panel) levels in HCC78 WT or ROS1 inhibitor resistant cells, treated for 3 days with the AKT inhibitor MK2206; significantly different from control (* p< 0.05; ** p< 0.01).

Article Snippet: MAPK inhibitor (U0126) was purchased from Promega and the AKT inhibitor (Mk2206) from Merck (Whitehouse Station, NJ, USA).

Techniques: Transduction, Plasmid Preparation, Expressing, Construct