Journal: PLoS ONE

Article Title: The Effects of the Endocannabinoids Anandamide and 2-Arachidonoylglycerol on Human Osteoblast Proliferation and Differentiation

doi: 10.1371/journal.pone.0136546

Figure Lengend Snippet: (A) RT-PCR analysis of cannabinoid receptor mRNA expression in HOBs. Human astrocytes (HA) were used as a positive control for CB1R, CB2R and TRPV1 expression. Hypoxanthine-guanine PhosphoRibosylTransferase (HPRT) was used as the control house-keeping gene. (B) ALP production, normalised to DNA content, was measured in HOBs grown for 4 days and treated with 10μM anandamide (AEA) only, or 10μM anandamide and the following antagonists and inhibitors. Cells were treated with AM251 (100nM, CB 1 R antagonist), AM630 (100nM, CB 2 R antagonist) or Capsazepine (1μM, Cap, TRPV1 antagonist) for 30 minutes before the addition of vehicle or anandamide. (C) Cells were treated with KT5720 (500nM, PKA inhibitor), PD98059 (1μM, MEK inhibitor) or SP600125 (10μm, JNK inhibitor) for 30 minutes before the addition of vehicle or anandamide. Data given as mean ± S.E.M., n = 9, from 3 experiments. * P <0.05, ** P <0.01, compared to antagonist/inhibitor only; one-way ANOVA with Sidak’s multiple comparisons test.

Article Snippet: HOBs were treated with media supplemented with 100nM AM251 (CB 1 R antagonist), 100nM AM630 (CB 2 R antagonist), 1μM capsazepine (TRPV1 antagonist), 500nM KT5720 (PKA inhibitor, PKA is upstream of CREB), 1μM PD98059 (MEK inhibitor) or 10μm SP600125 (JNK inhibitor) for 30 minutes before the addition of vehicle (0.1% ethanol) or 10μM anandamide (all Tocris, UK).

Techniques: Reverse Transcription Polymerase Chain Reaction, Expressing, Positive Control, Control

Journal: Molecular cell

Article Title: Irisin acts through its integrin receptor in a two-step process involving extracellular Hsp90α

doi: 10.1016/j.molcel.2023.05.008

Figure Lengend Snippet: Figure 5C

Article Snippet: # 9104S; RRID:AB_490881Rabbit Monoclonal Anti-Integrin αV (D2N5H)Cell Signaling TechnologyCat.

Techniques: Control, Virus, Recombinant, Expressing, Protease Inhibitor, Lysis, Transfection, Electron Microscopy, Immunodepletion, Clone Assay, Protein Purification, Endotoxin Assay, Bradford Assay, Staining, Mass Spectrometry, Mutagenesis, Software, Isolation

Journal: PLoS Genetics

Article Title: A high-throughput CRISPR interference screen for dissecting functional regulators of GPCR/cAMP signaling

doi: 10.1371/journal.pgen.1009103

Figure Lengend Snippet: (A) Schematic representation of the CREB reporter used in the CRISPR-based screen. Two cAMP response elements were fused to a ProteoTuner destabilizing domain (degron) followed by a green fluorescent protein, and inserted into a lentiviral vector. A clonal cell line was generated by transducing HEK293 cells with the reporter and dCas9-BFP-KRAB, sorting individual cells, and growing and verifying clonal lines for high reporter expression and efficient dCas9-dependent gene silencing. (B) The CREB reporter responds robustly to direct stimulation of adenylyl cyclase/cAMP signaling with forskolin. Reporter cells were treated with 10 μM forskolin (FSK) or DMSO (vehicle), and 1 μM Shield-1 was added simultaneously to stabilize the degron domain. After 4 h, reporter expression was analyzed by flow cytometry. Data from n = 4 per condition. (C) The CREB and PKA inhibitors, 666–15 and H89, respectively, significantly diminish FSK-induced accumulation of the reporter. Cells were pre-incubated with 100 nM 666–15 or 10 μM H89 versus DMSO (vehicle) for 30 min, then treated with 10 μM FSK in the presence of 1 μM Shield-1. After 4 h, reporter accumulation was analyzed by flow cytometry. Data plotted are from n = 5 for 666–15 and n = 7 for H89. (D) Timecourse for β2-AR-dependent reporter induction. Reporter cells were treated with 1 μM isoproterenol / 1 μM Shield-1 for indicated times or treated with 1 μM Shield-1 alone (no isoproterenol) for 5 h (“untreated”), and reporter expression was analyzed by flow cytometry. Data from n = 5. (E) Dose-response curve for isoproterenol-dependent reporter expression. Reporter cells were treated with indicated doses of isoproterenol and 1 μM Shield-1 for 4 h, and reporter expression was analyzed by flow cytometry. Data plotted are means of GFP expression, n = 3 per condition. EC 50 curve-fitting was performed using Prism6 GraphPad software. In each flow cytometry experiment, 10,000 cells total were analyzed and gated for singlets. Error bars = ± s.e.m. **** = p ≤ 0.0001; ** = p ≤ 0.01 by unpaired two-tailed Student’s t-test.

Article Snippet: The CREB inhibitor compound, 666–15, was purchased from Tocris Bioscience (Cat #5661), resuspended in DMSO and used at 100 nM final.

Techniques: CRISPR, Plasmid Preparation, Generated, Expressing, Flow Cytometry, Incubation, Software, Two Tailed Test