Ym 254890

Oleuropein (purity ≥ 80%, HPLC) for EA synthesis was from Shaanxi Huike Botanical Development Co. (Xi’an, China); metformin was from Cayman Chemicals (Ann Arbor, MI, USA); U73122 was from Tocris Bioscience (Pittsburgh, PA); YM 254,890 was from Focus Biomolecules (Plymouth Meeting, PA, USA); DMEM media, fetal bovine serum (FBS), and other cell culture supplements were from Hyclone (GE Healthcare Bio-Sciences, Pittsburgh, PA, USA); fluro-4AM was from ThermoFisher Scientific (Waltham, MA); antibodies for western blotting were from Santa Cruz Biotechnology (Dallas, TX, USA); assay kits for measuring active GLP-1 [GLP-1 (7–36) amide and GLP-1 (7–37)], triglyceride, human PYY as well as dipeptidyl peptidase IV (DPP4) inhibitor assay kit were from Cayman Chemical (Ann Arbor, MI); oleacein, hydroxytyrosol, oleocanthal, tyrosol, and oleuropein (≥ 95% pure) for GLP-1 secretion assays, RPMI1640 medium, and all other chemicals were purchased from Millipore Sigma (Burlington, MA, USA); IP₃ ELISA kit was from Amsbio (Cambridge, MA, USA); mouse insulin and leptin ELISA kits and mouse total GLP-1 ELISA kits were from Crystal Chem (Elk Grove Village, IL); glucose meter and strips were from AgaMatrix (Salem, NH); and free fatty acid (FFA) assay kits were from BioAssay Systems (Hayward, CA, USA).

IGF-II signal transduction may occur through different pathways (Figure 5a). We determined which of these could be associated with senescence. In particular, the MSC cultures were incubated at 37°C for 30 min with each of the following drugs, separately: 100 nM Pertussis toxin (PTX, BMLG101-0050 Enzo Biochem, NY, USA) for the Gαi kinase, 1 μM YM-254890 (YM, 10–1590 Focus Biomolecules, PA, USA) to block Gα_q/11, 50 μM D609 (sc201403, Santa Cruz Biotechology, CA, USA) to inhibit PLCβ, 10 μM BAPTA-AM (BPT) (15551 Cayman, MI, USA) and 10 mM 1,2,3,4-tetrahydrostaurosporine (STP, ab143861, Abcam, UK) for PKCα kinase inhibition, 5 nM PKCβi (Santa Cruz Biotech, TX, USA) for PKCβ inhibition, and 100 mM chlorpromazine (CPZ, sc-357313, Santa Cruz) to block endocytosis. Subsequently, we added 25 ng/ml IGF-II, and the samples were further incubated for 24 hr, and the senescence assays were performed. For each drug, we evaluated the inhibitory effect at the concentration we used (Figure 7—figure supplement 1).