Journal: Biochemistry

Article Title: Enzymatic Characterization of In Vitro Activity of RNA Methyltransferase PCIF1 on DNA

doi: 10.1021/acs.biochem.2c00134

Figure Lengend Snippet: Comparison of PCIF1 and MettL3-MettL14 activities on the same DNA substrate. (A, B) Comparison of activities of three enzymes (MettL3-MettL14, PCIF1, and HemK2-Trm112). Panel (B) is an enlarged portion of panel A in order to visualize the low activity of PCIF1. (C) Inhibition of MettL3-MettL14. The compounds tested were known inhibitors of MettL3, and included sinefungin, 5′-deoxy-5′-methylthioadenosine (MTA), the two racemates UZH1( R ) and UZH1( S ), and STM2457. (D) The MettL3-selective inhibitors had no effect on PCIF1. Compounds were used at a concentration of [I] = 50 μM.

Article Snippet: MettL3 inhibitors, two racemates of UZH1( R ) and UZH1( S ), and STM2457 were purchased from MCE (MedChemExpress).

Techniques: Comparison, Activity Assay, Inhibition, Concentration Assay

Journal: Endocrinology and Metabolism

Article Title: AM1638, a GPR40-Full Agonist, Inhibited Palmitate-Induced ROS Production and Endoplasmic Reticulum Stress, Enhancing HUVEC Viability in an NRF2-Dependent Manner

doi: 10.3803/EnM.2023.1774

Figure Lengend Snippet: Nuclear factor erythroid 2–related factor 2 (NRF2)-mediated signaling was activated after AM1638 (AM) treatment but not after TAK875 (TAK) or LY2922470 (LY) treatment. Human umbilical vein endothelial cells (HUVECs) were stimulated with the indicated G protein-coupled receptor 40 (GPR40) agonists (20 μM) for 24 hours. (A) Kelch-like ECH-associated protein 1 (Keap1), NRF2, and nuclear NRF2 levels were determined by Western blotting. (B) Western blotting shows the intracellular abundance of heme oxygenase-1 (HO-1) and nicotinamide adenine dinucleotide phosphate: quinone oxidoreductase 1 (NQO1). (C) GPR40 level was limited by small interfering RNA (siRNA). (D, E) In HUVECs transfected with scrambled (Scr) or GPR40 siRNA, nuclear NRF2 level and HO-1 and NQO1 expression were detected by Western blotting. Each mean±standard deviation was obtained from three separate experiments. Veh, vehicle; W.E., whole cell extract; N.E., nuclear extract. a P <0.05 vs. the Veh group; b P <0.05 vs. the AM group; as analyzed by analysis of variance (ANOVA) followed by the Tukey-Kramer test.

Article Snippet: The GPR40-partial agonists LY2922470 and TAK875 (MedChemExpress, Monmouth Junction, NJ, USA), GPR40-full agonist AM1638 (MedChemExpress), and the NRF2 inhibitor ML385 (Sigma Aldrich, St. Louis, MO, USA) were dissolved in dimethyl sulfoxide (Sigma Aldrich).

Techniques: Western Blot, Small Interfering RNA, Transfection, Expressing, Standard Deviation

Journal: Endocrinology and Metabolism

Article Title: AM1638, a GPR40-Full Agonist, Inhibited Palmitate-Induced ROS Production and Endoplasmic Reticulum Stress, Enhancing HUVEC Viability in an NRF2-Dependent Manner

doi: 10.3803/EnM.2023.1774

Figure Lengend Snippet: Palmitate-induced superoxide production was reduced after AM1638 (AM) treatment but not after TAK875 (TAK) or LY2922470 (LY) treatment. Human umbilical vein endothelial cells (HUVECs) were pretreated with the indicated chemicals for 24 hours and then stimulated with palmitate (400 μM) for 6 hours. The cells were stained with dihydroethidium (DHE). (A, C) The superoxide levels were visualized using an immunofluorescence microscope (×200). The levels of red fluorescence were obtained using ImageJ software and then normalized to the number of cells. (B, D) The mean fluorescence intensity (MFI) for red fluorescence was calculated by flow cytometry. Each mean±standard deviation was obtained from three separate experiments. Veh, vehicle; PA, palmitate; ML, ML385. a P <0.05 vs. the Veh group; b P <0.05 vs. the PA group; c P <0.05 vs. the PA plus AM group, as analyzed by analysis of variance (ANOVA) followed by the Tukey-Kramer test.

Article Snippet: The GPR40-partial agonists LY2922470 and TAK875 (MedChemExpress, Monmouth Junction, NJ, USA), GPR40-full agonist AM1638 (MedChemExpress), and the NRF2 inhibitor ML385 (Sigma Aldrich, St. Louis, MO, USA) were dissolved in dimethyl sulfoxide (Sigma Aldrich).

Techniques: Staining, Immunofluorescence, Microscopy, Fluorescence, Software, Flow Cytometry, Standard Deviation

Journal: Endocrinology and Metabolism

Article Title: AM1638, a GPR40-Full Agonist, Inhibited Palmitate-Induced ROS Production and Endoplasmic Reticulum Stress, Enhancing HUVEC Viability in an NRF2-Dependent Manner

doi: 10.3803/EnM.2023.1774

Figure Lengend Snippet: Palmitate-induced endoplasmic reticulum (ER) stress was inhibited after treatment with AM1638 (AM), TAK875 (TAK), or LY29-22470 (LY). (A, B, C) Human umbilical vein endothelial cells (HUVECs) were pretreated with the indicated G protein-coupled receptor 40 (GPR40) agonist (20 μM) for 24 hours and then stimulated with palmitate (400 μM) for 6 hours. Western blotting showed the intracellular levels of Kelch-like ECH-associated protein 1 (Keap1), heme oxygenase-1 (HO-1), protein kinase R-like endoplasmic reticulum kinase (PERK) phosphorylation, CCAAT/enhancer‐binding protein homologous protein (CHOP), inositol requiring enzyme 1α (IRE1α) phosphorylation, and spliced X-box binding protein-1 (XBP-1s). (D, E, F) HUVECs were pretreated with AM1638 (20 μM) or AM1638 plus ML385 (2 or 5 μM) for 24 hours and then incubated with palmitate (400 μM) for 6 hours. The levels of the indicated proteins were determined by Western blotting. Each mean±standard deviation was obtained from three separate experiments. Veh, vehicle; PA, palmitate; ML, ML385. a P <0.05 vs. the Veh group; b P <0.05 vs. the PA group; c P <0.05 vs. the PA plus AM group, as analyzed by analysis of variance (ANOVA) followed by the Tukey-Kramer test.

Article Snippet: The GPR40-partial agonists LY2922470 and TAK875 (MedChemExpress, Monmouth Junction, NJ, USA), GPR40-full agonist AM1638 (MedChemExpress), and the NRF2 inhibitor ML385 (Sigma Aldrich, St. Louis, MO, USA) were dissolved in dimethyl sulfoxide (Sigma Aldrich).

Techniques: Western Blot, Binding Assay, Incubation, Standard Deviation

Journal: Endocrinology and Metabolism

Article Title: AM1638, a GPR40-Full Agonist, Inhibited Palmitate-Induced ROS Production and Endoplasmic Reticulum Stress, Enhancing HUVEC Viability in an NRF2-Dependent Manner

doi: 10.3803/EnM.2023.1774

Figure Lengend Snippet: Palmitate-induced cell death was blocked after treatment with AM1638 (AM), TAK875 (TAK), or LY2922470 (LY). Human umbilical vein endothelial cells (HUVECs) were pretreated with the indicated chemicals for 24 hours and then stimulated with palmitate (400 μM) for 24 hours. (A, C) Western blotting showed the cleaved caspase-3 and poly(ADP-ribose) polymerase (PARP) levels. (B, D) Cell viability was measured using EZ-CYTOX solution (Daeil Lab Service). Each mean±standard deviation was obtained from three separate experiments. Veh, vehicle; PA, palmitate; ML, ML385; OD, optical density. a P <0.05 vs. the Veh group; b P <0.05 vs. the PA group; c P <0.05 vs. the PA plus AM group, as analyzed by analysis of variance (ANOVA) followed by the Tukey-Kramer test.

Article Snippet: The GPR40-partial agonists LY2922470 and TAK875 (MedChemExpress, Monmouth Junction, NJ, USA), GPR40-full agonist AM1638 (MedChemExpress), and the NRF2 inhibitor ML385 (Sigma Aldrich, St. Louis, MO, USA) were dissolved in dimethyl sulfoxide (Sigma Aldrich).

Techniques: Western Blot, Standard Deviation

Journal: Endocrinology and Metabolism

Article Title: AM1638, a GPR40-Full Agonist, Inhibited Palmitate-Induced ROS Production and Endoplasmic Reticulum Stress, Enhancing HUVEC Viability in an NRF2-Dependent Manner

doi: 10.3803/EnM.2023.1774

Figure Lengend Snippet: Palmitate-induced nuclear damage was reduced after treatment with AM1638 (AM), TAK875 (TAK), or LY2922470 (LY). Cells were pre-incubated with the indicated chemicals for 24 hours and then stimulated with palmitate (400 μM) for 24 hours. (A, C) Human umbilical vein endothelial cells (HUVECs) were stained with Hoechst to observe the nuclear morphology under a fluorescence microscope (×200). The white arrows point to cells whose nucleus changed. (B, D) Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL)-positive and -negative HUVECs were counted under a fluorescence microscope (×200). The white arrows indicate nuclearcleaved cells. Each mean±standard deviation was obtained from three separate experiments. Veh, vehicle; PA, palmitate; ML, ML385; DAPI, 4´,6-diamidino-2-phenylindole. a P <0.05 vs. the Veh group; b P <0.05 vs. the PA group; c P <0.05 vs. the PA plus AM group, as analyzed by analysis of variance (ANOVA) followed by the Tukey-Kramer test.

Article Snippet: The GPR40-partial agonists LY2922470 and TAK875 (MedChemExpress, Monmouth Junction, NJ, USA), GPR40-full agonist AM1638 (MedChemExpress), and the NRF2 inhibitor ML385 (Sigma Aldrich, St. Louis, MO, USA) were dissolved in dimethyl sulfoxide (Sigma Aldrich).

Techniques: Incubation, Staining, Fluorescence, Microscopy, TUNEL Assay, Standard Deviation

Journal: Endocrinology and Metabolism

Article Title: AM1638, a GPR40-Full Agonist, Inhibited Palmitate-Induced ROS Production and Endoplasmic Reticulum Stress, Enhancing HUVEC Viability in an NRF2-Dependent Manner

doi: 10.3803/EnM.2023.1774

Figure Lengend Snippet: The actions of G protein-coupled receptor 40 (GPR40)-specific agonists in human umbilical vein endothelial cells. AM1638 (AM), a GPR40-full agonist, can activate nuclear factor erythroid 2–related factor 2 (NRF2)-related pathways and reduce palmitate-induced superoxide production, enhancing cell viability. However, GPR40-partial agonists, TAK875 (TAK) and LY2922470 (LY), can decrease palmitate-induced cytotoxicity without blocking superoxide production or activating NRF2 pathways. HO-1, heme oxygenase-1; NQO1, nicotinamide adenine dinucleotide phosphate: quinone oxidoreductase 1; ER, endoplasmic reticulum; PERK, protein kinase R-like endoplasmic reticulum kinase; CHOP, CCAAT/enhancer‐binding protein homologous protein; IRE1α, inositol requiring enzyme 1α; XBP-1, X-box binding protein-1; PARP, poly (ADP-ribose) polymerase.

Article Snippet: The GPR40-partial agonists LY2922470 and TAK875 (MedChemExpress, Monmouth Junction, NJ, USA), GPR40-full agonist AM1638 (MedChemExpress), and the NRF2 inhibitor ML385 (Sigma Aldrich, St. Louis, MO, USA) were dissolved in dimethyl sulfoxide (Sigma Aldrich).

Techniques: Blocking Assay, Binding Assay