Journal: Scientific Reports

Article Title: Tectorigenin induces vasorelaxation in porcine coronary arteries through activation of Kv channels and oestrogen receptor modulation

doi: 10.1038/s41598-025-20988-6

Figure Lengend Snippet: Effects of neural and cyclic nucleotide pathway inhibitors on tectorigenin-induced relaxation in porcine coronary arteries pre-contracted with 100 nM U46619. ( A ) Tetrodotoxin (TTX, 1 µM) and ω-conotoxin GVIA (CTX, 1 µM) had no significant effect on the relaxation induced by 30 µM tectorigenin ( p > 0.05, n = 4). ( B ) Pretreatment with rolipram (1 µM, a selective phosphodiesterase-4 inhibitor) or vardenafil (1 µM, a selective phosphodiesterase-5 inhibitor) did not significantly alter tectorigenin-induced vasorelaxation ( p > 0.05, n = 4). ( C ) Inhibitors of the nitric oxide and cyclic nucleotide pathways, including Nω-nitro-L-arginine (L-NNA, 100 µM), KT5720 (1 µM, a PKA inhibitor), and KT5823 (1 µM, a PKG inhibitor), also did not significantly affect the relaxant response to tectorigenin ( p > 0.05, n = 4). Data are expressed as mean ± standard error of the mean (SEM) from four independent hearts. U46619 plateau (normalised to 60 mM KCl) was similar across groups ( p > 0.05; Supplementary Table 2).

Article Snippet: For experimental assays, a range of pharmacological agents was utilised, including U46619, apamin, KT5720, KT5823, and L-NNA (Sigma-Aldrich, MO, USA); rolipram, vardenafil, and TEA (Santa Cruz Biotechnology, CA, USA); IbTX (Alomone Labs, Jerusalem, Israel); glibenclamide (Research Biochemicals International, MA, USA); TTX and 4-AP (Tocris Bioscience, Bristol, UK); CTX (Bachem, Bubendorf, Switzerland); and charybdotoxin, methyl-piperidino-pyrazole (MPP), and PHTPP (Cayman Chemical, MI, USA).

Techniques:

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: Cells

Article Title: Inhibition of Vascular Smooth Muscle Cell Proliferation by ENPP1: The Role of CD73 and the Adenosine Signaling Axis.

doi: 10.3390/cells13131128

Figure Lengend Snippet: Figure 6. Effect of AMP on activation of Gs-coupled adenosine receptors and PKA. Synthetic VSMCs were pretreated for 18 h with either (a) dual A2aR/A2bR antagonist AB928 (0.3 µM) or (b) selective A2aR antagonist SCH-58261 (1 µM) and selective A2bR antagonist PSB-1115 (1 µM). Cells were washed and stimulated for 4 h with AMP in the presence or absence of inhibitors. Total cAMP was measured using HTRF assay. VSMCs were preincubated for 30 min in the basal media supplemented with 0.25% FBS and stimulated for 30 min with AMP in the absence (c) or presence (d) of PKA inhibitor KT5720. Phosphorylation of VASP at Ser157 was analyzed using HTRF assay. Values are presented as the mean ± SD, n = 4–6, ** p < 0.01, *** p < 0.001 (t-test).

Article Snippet: ATP, PKA inhibitor KT5720, A2aR antagonist SCH-58261, and A2bR antagonist PBS-1115 were from Tocris Biosciences (Minneapolis, MN, USA).

Techniques: Activation Assay, HTRF Assay, Phospho-proteomics

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: The American Journal of Pathology

Article Title: A Role for cAMP and Protein Kinase A in Experimental Necrotizing Enterocolitis

doi: 10.1016/j.ajpath.2016.10.014

Figure Lengend Snippet: A: PKA inhibitor (KT5720) inhibits caspase-3 activation and PKA activation in vitro. After 4 to 6 hours of infection both caspase-3 activation and pPKA were significantly increased. IEC-6 cells were treated with 1 μmol/L of KT5720 before exposure to CS demonstrated significantly less caspase-3 and PKA activation after 6 hours. B: CS-induced apoptosis is decreased by pharmacologic PKA inhibition. Immunofluorescence of DNA fragmentation and apoptosis were significantly decreased in the presence of CS even after 6 hours of infection when cells were pretreated with PKA inhibitors (KT5720, Rp-8-br-cAMPS, PKI) compared with those cells that received CS alone. The greatest decrease occurred in those cells treated with KT5720 compared with other inhibitors. IEC-6 cells were grown on chamber slides, and after various doses and exposure times they were fixed and stained with ApoTag for apoptosis and DAPI (blue). Apoptotic cells were counted per 1000 cells. C: Knockdown of PKA showed a significant reduction in caspase-3 cleavage. IEC-6 cells were transfected with lipofectamine 2000 and siRNA for PKA. Cells were then treated for 6 hours with CS. Western blot analysis of cleaved caspase-3, PKA, and β-actin were performed. PKA knockdown was confirmed with a significant reduction in PKA expression. A significant reduction in cleaved caspase 3 was also seen at the 6-hour time point. D: Knockdown of PKA protects IEC-6 cells from CS-induced apoptosis. IEC-6 cells were transfected with lipofectamine 2000 and siRNA for PKA. Cells were then treated over a time course with CS. There was a significant increase in apoptosis in the presence of CS after 6 hours. In addition, ApoTag staining revealed fewer apoptotic cells when PKA was knocked down (+siRNA) before infection with CS for 6 hours than controls (−siRNA) (P = 0.0014). ∗P < 0.05, ∗∗∗P < 0.001, and ∗∗∗P < 0.001, ∗∗∗∗P < 0.0001. Original magnification, ×10 (B and D). CS, Cronobacter sakazakii; IEC-6, rat intestinal epithelial cell line; KD, knockdown; PKA, protein kinase A; PKI, protein kinase inhibitor; pPKA, PKA phosphorylation: WT, wild-type.

Article Snippet: Cells were pretreated with PKA inhibitors (0.1–20 μmol/L) KT5720 (Cayman Chemical, Ann Arbor, MI), cAMP Dependent Protein Kinase Inhibitor (Sigma-Aldrich, St. Louis, MO), Rp-8-cAMPS (Santa Cruz Biotechnology, Dallas, TX) for 30 minutes then subjected to co-culture with C. sakazakii at various concentrations over a time course.

Techniques: Activation Assay, In Vitro, Infection, Inhibition, Immunofluorescence, Staining, Knockdown, Transfection, Western Blot, Expressing, Phospho-proteomics

Journal:

Article Title: Functional differences between cardiac and renal isoforms of the rat Na + -Ca 2+ exchanger NCX1 expressed in Xenopus oocytes

doi: 10.1111/j.1469-7793.2000.00599.x

Figure Lengend Snippet: A, 45Ca2+ uptake over a 20 min period was measured in Na+-loaded oocytes expressing NCX1 after reducing [Na+]o to 0 mM. PKA activity was enhanced with a cocktail of IBMX, dibutyryl cAMP and forskolin. KT5720 (1 μM) was added to the cocktail where indicated (KT). 45Ca2+ uptake by the oocytes in the presence and absence of PKA cocktail was compared and 45Ca2+ uptake in PKA-treated oocytes in the presence and absence of KT5720 was also shown. Double asterisks indicate a significant difference, P < 0.01, n = 6. B, 45Ca2+ uptake for a period of 10 min after depolarizing the adult rat ventricular cardiomyocytes in 0 Na+, 144 mM K+. All solutions contained 100 μM ouabain and 10 μM nifedipine including solutions used for the 30 min pretreatment. Ni2+ (4 mM) was added with 0 Na+, 144 mM K+ solution in some experiments. The PKA cocktail (see A) was added in 0 Na+, 144 mM K+ solution, and in some experiments KT (1 μM) was also added. 45Ca2+ uptake by the cardiomyocytes in the presence and absence of Ni2+ was compared and 45Ca2+ uptake in PKA-treated cardiomyocytes in the presence and absence of KT5720 was also shown. Double asterisks indicate significant differences, P < 0.001 (n = 6). C, INaCa from oocyte-expressing cardiac Na+-Ca2+ exchanger before (▪) and during (at 5 and 10 min) the incubation with PKA-activating cocktail (▵, ▾). D, averaged results of the enhanced current of the cardiac Na+-Ca2+ exchanger at 1, 5 and 10 min after the PKA activation at three different voltages (-20, +20 and +60 mV). Data at 1 min at each voltage shown after PKA treatment are compared to data at 5 and 10 min. Asterisks denote significant differences (P < 0.001, n = 5). E, INaCa from oocytes expressing renal Na+-Ca2+ exchanger before and during activation of PKA as in panel C.

Article Snippet: The forskolin was dissolved in ethanol while A23187, IBMX and KT5720 (KT) (LC Laboratories, Wobum, MA, USA) were dissolved in DMSO.

Techniques: Expressing, Activity Assay, Incubation, Activation Assay