Dpp 4

Supernatants of TGFβ1-stimulated FBs after gene knockdown and treatment with protease inhibition were collected, centrifuged, and stored at −20 °C for further use. Protein levels of human procollagen Ia1 ELISA (R&D Systems) and human fibronectin ELISA (R&D Systems) were measured according to the manufacturer’s manual. Absorbance was detected by FluoStar Optima microplate reader (BMG Labtech, Ortenberg, Germany). Six-millimeter punch biopsies of healthy skin and hypertrophic scar tissue were lysed in 1% Triton X-100 lysis buffer (Sigma) and mechanically homogenized using precellyse tissue homogenizer. After centrifugation, lysates were analyzed using DPP4 and urokinase ELISAs (both R&D Systems) Total protein concentrations were measured using a BCA-kit (Abcam) according to the manufacturer’s protocol, and concentrations were normalized to total protein.

Plasma was used for biochemical measurements with reagents from Randox Laboratories Ltd. (County Antrim, UK). Plasma Glucose was measured by Glucose Oxidase method, Total Cholesterol by Cholesterol Oxidase method, Total Triglycerides by Lipase/GPO-PAP method. Glycated hemoglobin (HbA1c) was measured by HPLC (D10 Hemoglobin analyzer, Bio-Rad, Hercules, CA, USA). Plasma Leptin (RayBiotech, Norcross, GA, USA), Insulin (Merck Millipore, MA, USA), DPP4 (R&D Systems, MN, USA), and GLP-1(Active) (Merck Millipore, MA, USA) levels were measured by ELISA. Homeostatic model assessment (HOMA2) designed by Diabetes Trials Unit, The Oxford Center for Diabetes, Endocrinology, and Metabolism was used to estimate Insulin resistance (HOMA2 IR) from all fasting venous samples.

Immunoblotting was performed on mouse heart homogenates, isolated adult mouse cardiomyocytes or C166 endothelial cell lysates with the following antibodies: phosphorylated phospholamban (phospho-PLN) Ser16 1:1000 (A285) [42 (link)], total PLN 1:1000 (1D11) [43 (link)], DPP-4 1:1000 (AF954, R & D Systems, Minneapolis, MN, USA); phospho-Akt (Ser473) 1:1000 (#9271, Cell Signaling Technology, Danvers, MA, USA), total Akt 1:1000 (#9272, Cell Signaling Technology), phospho-endothelial nitric oxide synthase (phospho-eNOS) (Ser1177) 1:1000 (#07-428-I, Sigma-Aldrich), or GAPDH 1:1000 (#2118, Cell Signaling Technology). Densitometry was performed using ImageJ version 1.39 (National Institutes of Health, Bethesda, MD, USA).

The medium of the hMADS cells was collected over 24 h, from day 13 (after replacement of medium) to day 14 of differentiation, to determine the secretion of adipokines using high-sensitive ELISAs (leptin and DPP-4 from R&D Systems, Inc., Minneapolis, MN, USA; IL-6 and MCP-1 from Diaclone SAS, Besancon Cedex, France; adiponectin and PAI-1 from BioVendor–Laboratorni medicina a.s., Brno, Czech Republic). If necessary, samples were diluted with the dilution buffer provided by the manufacturer prior to the assay, which was performed in duplicates according to the manufacturer’s instructions.

The cell lysis buffer (Cell Signaling Technology, California, USA) containing protease and phosphatase inhibitor cocktail (Abcam, USA) was used for the extraction of total proteins from the chondrocytes, followed by quantification with a BCA kit (Shanghai Ze Ye Biotechnology Co., Ltd, Shanghai, China). After loading approximately 30 μg protein, 12% SDS PAGE was used to separate the proteins, which were further transferred to the PVDF membrane (Cell Signaling Technology, California, USA). Then, the protein-loaded PVDF membrane was mixed with 5% skim milk to block the nonspecific binding proteins, followed by incubation in the solution of primary antibody against SOX-9 (1:1000, R&D, Minnesota, USA), Nrf2 (1:2000, R&D, Minnesota, USA), DPP-4 (1:2500, R&D, Minnesota, USA), Lamin B1 (1:500, R&D, Minnesota, USA) and GAPDH (1:800, R&D, Minnesota, USA). The membrane was subsequently incubated with the secondary antibody (R&D, Minnesota, USA). Lastly, the bands were visualized using the enhanced chemiluminescence (ECL) kits (Cell Signaling Technology, California, USA) on a Tanon-2500 imaging system (Shanghai, China), followed by quantifying the relative expression level of target proteins with Image J software ²⁵.

To evaluate cell proliferation of rat aortic smooth muscle cells, the bromodeoxyuridine (BrdU) incorporation assay was performed using a Cell Proliferation ELISA kit (1647229; Roche Applied Science, Penzberg, Germany), as previously described [18 (link),19 (link)]. Briefly, rat aortic smooth muscle cells were plated at 2000 cells/well in 96-well culture plates in complete media (n =5), and were incubated in Dulbecco’s Modified Eagle Medium (DMEM) with 10% fetal bovine serum (FBS). After attaining 60–70% confluence, rat aortic smooth muscle cells were incubated in DMEM containing 0.1% FBS with or without 10 nM linagliptin for 48 h, and then stimulated with PDGF (25 ng/ml; Sigma-Aldrich, St Louis, MO, USA) or DPP-4 (200 ng/ml; R&D Systems) for 24 h. BrdU solution (10 μM) was added during the last 2 h of stimulation. Next, the cells were dried and fixed, and cellular DNA was denatured with FixDenat solution (Roche Applied Science) for 30 min at room temperature. A peroxidase-conjugated, mouse anti-BrdU monoclonal antibody (Roche Applied Science) was added to the culture plates and incubated for 90 min at room temperature. Finally, tetramethylbenzidine substrate was added for 15 min at room temperature and absorbance of the samples was measured using a microplate reader at 450–620 nm. Mean data are expressed as a ratio of control (non-treated) cell proliferation.