Cariporide

Tamoxifen, DAPI (4’,6-Diamidino-2-Phenylindole, Dihydrochloride), Cariporide (HOE642), Thapsigargin, Lucigenin, NADPH, Superoxide Dismutase from bovine erythrocytes (SOD), Diphenyleneiodonium chloride (DPI), 2’-7’-dichlorofluorescein diacetate (DCFH-DA) and Centricon Plus-70 Centrifugal Filter were from Sigma-Aldrich (St. Louis, MO, USA). Amicon Ultra-4 Centrifugal Filter Unit and GSK2606414 was from Millipore (Billerica, MA, USA). iTaq™ Universal SYBR® Green, and iScript cDNA kit, were from Bio-rad laboratories (Hercules, CA, USA). GKT137831 was from Selleckchem (Houston, TX, USA). Direct-zol™ RNA MicroPrep was from Zymo Research (Irvine, CA, USA). Adult Brain Dissociation Kit, mouse and rat, Anti-ACSA-2 MicroBead Kit were from Miltenyi Biotec (Germany).

Transmigration was determined employing Boyden chamber assays. 20 µl of the collagen I mixture (composition as described above) were allowed to polymerize on a filter-membrane (insert for a 24 well plate, 8.0 µm pore size; ThinCert, Greiner Bio-One GmbH) at 37 °C in a humidified atmosphere overnight. 200,000 cells per filter were seeded onto this collagen matrix. After 24 h incubation in RPMI1640 with G-418 and serum, the medium was gently renewed for another 24 h. Cells were then fixed and stained with crystal violet (Sigma-Aldrich) in PBS. The matrix and the remaining cells on the upper side of the filter were removed and excess crystal violet was washed away with PBS. The invasive cells that remained on the lower side of the filter and those on the bottom of the well were counted.
MMP activity was inhibited by 10 µmol l⁻¹ NNGH (N-isobutyl-N-(4-methoxyphenylsulfonyl)glycyl hydroxamic acid; Sigma-Aldrich), and NHE1 was inhibited with 10 µmol l⁻¹ cariporide (HOE642). DMSO, the solvent for NNGH and cariporide, reached a final concentration of 0.1%.
Total MMP activity was disabled by fixing the matrix with 1 ml of 2% glutaraldehyde in PBS (v/v) for 15 min. In order to ascertain the role of actin in transmigration through a fixed matrix, cells were exposed to Cytochalasin D (50 nmol l⁻¹) over the entire experiment (48 h).

Murine microvascular SVEC4-10EE2 endothelial cell line cells (EE2 ECs; ATCC, Manassas, VA) were cultured as described earlier [22 ]. Previously, MG-sensitive SGK1 signaling was studied in EE2 ECs [22 ] and in the present study we elucidate the role of NHE1 in MG-induced leukocyte recruitment in the context of SGK1-dependent activation of NHE1. We, therefore, used EE2 ECs to corroborate our in vivo findings. Where indicated, Tempol (300 μM; Sigma), cariporide (50 μM) or GSK650394 (20 μM; Sigma) was added at the specified concentrations. Targeted gene silencing was accomplished by a 48-h transfection of EE2 ECs with siRNA specifically targeting SGK (Santa Cruz) and with siRNA transfection medium and reagent (Santa Cruz) as described previously [48 (link)]. The control cells were transfected with negative control scrambled siRNA (Santa Cruz) having no homology to any known RNA sequence.

Cells were treated with cariporide (Sigma-Aldrich, Inc., St. Louis, MO, USA, stock in 200 μg/ml in DMSO). Cells were trypsinized and transferred into a 96-well plate at a density of 2 × 10³ cells per well, and incubated with drugs at different concentrations of cariporide (3–100 μg/ml), doxorubicin (1–100 μg/ml) and paclitaxel (0.2–30 μg/ml) for 24 h, 48 h. CCK-8 solution (Beyotime Biotechology, Nantong, China, 10 μl in 100 μl of medium) was added to each well, and the cells were incubated for 2 h at 37 °C. The optical density value at 450 nm was determined using a microplate reader (Imark, Lab system; Bio-Rad, USA).

Cariporide, N-acetylcysteine (NAC), Bovine serum albumin (BSA, cat. A1933, reagent ≥ 98%), and D-glucose were purchased from Sigma Company. Antibody to AGEs receptor (Ab-RAGE) was purchase from Santa Cruz Company. BCA protein assay kit was brought from PIECE Company.