Fixdenat solution

To evaluate LNCaP cell proliferation, the bromodeoxyuridine (BrdU) incorporation assay was performed using Cell Proliferation ELISA kits (1647229; Roche Applied Science, Mannheim, Germany) as described previously [8 (link)]. Briefly, LNCaP cells with or without siRNA transfection were plated at 5000 cells/well in 96-well culture plates in complete media. After attaining 60–70% confluence, LNCaP cells were treated with or without 10 nM Ex–4, 0.1 mM metformin, a combination of 10 nM Ex–4 and 0.1 mM metformin, or Compound C in media with 10% FBS for 24 h. BrdU solution (10 μM) was added during the last 2 h of stimulation. Next, the cells were dried and fixed, and the cellular DNA was denatured with FixDenat solution (Roche Applied Science) for 30 min at room temperature (RT). A peroxidase-conjugated mouse anti-BrdU monoclonal antibody (Roche Applied Science) was added to the culture plates and incubated for 90 min at RT. Finally, tetramethylbenzidine substrate was added for 5 min at RT and the absorbance of the samples was measured using a microplate reader (Thermo Fisher Scientific K.K., Yokohama, Japan) at 450–620 nm. Mean data are expressed as a ratio of the control (untreated) cell proliferation.

In order to measure proliferation rate, Bromodeoxyuridine (BrdU) incorporation assay was carried out using a cell proliferation ELISA BrdU kit (Roche Diagnostics, USA) according to manufacturer's instructions. Briefly, UMR-106 cells were seeded at 2500 cells/well in 96-well plates in FBS-supplemented media. After 24 h of incubation, cells were labeled with 10 µM BrdU and incubated for an additional 4 h at 37°C. Labeling medium was removed from the microplates, cells were dried and fixed, and cellular DNA was denatured with FixDenat solution (Roche Diagnostics, USA) for 30 min at room temperature. A mouse peroxidase-conjugated anti-BrdU monoclonal antibody was added to each well and plates were incubated again at room temperature for 2 h. After plates were washed, they were incubated with substrate solution containing hydrogen peroxide, luminol and 4-iodophenol. The immunocomplex was quantified by luminescence emission in a luminometer Fluoroskan Ascent FL (Labsystems). The assay was performed in triplicates and results were expressed as cell proliferation percentage, taking control cells as 100% proliferation ± SEM.

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.

To evaluate the cell proliferation of RASMCs, a bromodeoxyuridine (BrdU) incorporation assay was performed using a Cell Proliferation ELISA kit (1647229; Roche Applied Science, Mannheim, Germany). Briefly, RASMC were seeded at 4000 cells/well in 96-well culture plates in complete media (n = 5). At 60%–70% confluence, cells were treated with 10 nM linagliptin, 500 nM empagliflozin, or both for 48 h. A BrdU (10 μM) solution was added during the last 2 h of treatment. Next, the cells were dried and fixed, and the 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, followed by incubation for 90 min at room temperature. Finally, tetramethylbenzidine substrate was applied for 15 min at room temperature, and the absorbance of the samples was measured using a microplate reader at 450–620 nm. Mean data are expressed as a ratio of the control (untreated) cell proliferation.