Egm 2 mv singlequots

HUVECs and HDMVECs purchased from ATCC were cultured in EBM supplemented with EGM-2MV singleQuots (Lonza) without hydrocortisone in 0.2% gelatin-coated tissue culture plates. All experiments were performed using HUVECs of passage 6 or lower. Recombinant histone H4 was purchased from Cayman (catalog 10264) for in vitro experiments. Histone from calf thymus was purchased from MilliporeSigma (catalog 10223565001). Anti–histone H4 was from Cell Signaling Technology (catalog 2592). TLR2 inhibitor C29 was from Medchemexpress (catalog HY-100461), and TLR4 inhibitor TAK 242 was from MilliporeSigma (catalog 614316). Citrullinated histone H4 (catalog 17927), SCH 58261 (catalog 19676), PSB 603 (catalog 25637), and wortmannin (catalog 10010591) were purchased from Cayman.

The human ccRCC cell line Caki-2 was obtained from Sigma-Aldrich, St. Louis, MO, USA (cat. no. 93120819). Caki-1 cells were obtained from ATCC (cat no. HTB-46, Manassas, VA, USA). HUVECs were kindly provided by the Department of Transplantology, Medical College (Krakow, Poland). The cells in the initial vials were expanded and cryopreserved, and cells were propagated with less than fifteen consecutive passages. All cell lines were routinely tested for mycoplasma by PCR every three months. The Caki-2 and Caki-1 cell lines were cultured in Eagle’s minimal essential medium (EMEM; Lonza, Basel, Switzerland) supplemented with 10% fetal bovine serum (FBS). HUVECs were cultured in endothelial basal medium (EBM-2, Lonza) supplemented with EGM-2 MV SingleQuots (Lonza). All cell lines were cultured at 37 °C in a humidified incubator in a 5% CO₂ atmosphere. To collect conditioned medium from the Caki-1 and Caki-2 cell lines, cells were cultured for 7 days in the presence of drugs, followed by 24 h in medium supplemented with only 0.5% BSA (BioShop, Burlington, Ontario, Canada); the culture medium was then collected for further analysis.

The cord blood derived CD34+ HPCs were plated on fibronectin-coated tissue culture flasks and cultured in endothelial basal medium (EBM-2, Lonza) supplemented with EGM-2-MV-SingleQuots (Lonza) and 1% penicillin-streptomycin (Sigma-Aldrich) in a humidified incubator at 37°C with 5% CO2. After 4 days of culture, nonadherent cells were discarded by washing with PBS. Medium were changed every 2 days thereafter. To confirm the EPC phenotype after culturing for 7 days, adherent cells were incubated with DiI-labeled Ac-LDL (Molecular Probes) for one hour before visualizing with an inverted fluorescent microscope (Leica DM IRE2). Immunostaining with antibodies against VEGFR2(Flk-1,1∶50 dilution; Santa Cruz Biotechnology), CD31(1∶100 dilution; Cell Signaling) followed by Cy3-conjugated goat anti-mouse antibody(1∶250 dilution; Invitrogen) was performed, and then cells were visualized with an inverted fluorescent microscope (Leica DM IRE2). After culturing for 14 days, cells were stained with anti-von Willebrand factor antibody FITC (1∶100 dilution; Abcam), or stained with antibodies against VEGFR2 (Flk-1,1∶50 dilution; Santa Cruz Biotechnology), CD31(1∶100 dilution; Cell Signaling) followed by Cy3-conjugated goat anti-mouse antibody(1∶250 dilution; Invitrogen), and then visualized with an inverted fluorescent microscope(Leica DM IRE2).

Human epidermal keratinocytes were isolated from neonatal foreskin as previously described [21] (link) and cultured in KBM-Gold basal medium (Lonza, Walkersville, MD) with growth supplements (KGM-Gold Single-Quot Kit, Lonza). Keratinocytes, no later than passage 4, were switched to 550 µM calcium 16–18 hours prior to experimental manipulation, unless other noted. For the experiments in Figure 4 and Figure 8B, keratinocytes were grown in 50 µM calcium to prevent desmosome assembly for 16–18 hours and then switched to 550 µM calcium for the indicated times. Chinese hamster ovary (CHO) cells were cultured in F12 media (ATCC, Manassas, VA). HMEC-1s were cultured in 0.1% gelatin-coated flasks in EBM-2 media (Lonza) with growth supplements (EGM-2 MV SingleQuots, Lonza). Where indicated, cells were infected 24–48 hours prior to experimentation with adenovirus for expression of Dsg3.GFP, as previously described [32] (link).

Human coronary artery endothelial cells (HCAECs), endothelial basal medium-2 (EBM-2), EGM-2-MV SingleQuots, fetal bovine serum (FBS), and phosphate-buffered saline (PBS) were purchased from Lonza (Viviere, Belgium). HCAECs were maintained in EBM-2 supplemented with EGM-2-MV SingleQuots (containing vascular endothelial growth factor, basic fibroblast growth factor, insulin-like growth factor-I, epidermal growth factor, ascorbic acid, and gentamicin), and 10% FBS. All cells were cultured at 37°C in an incubator with a humidified atmosphere and 5% CO₂. Cells were split at the ratio of 1:3 every passage. Cells from three to six passages were used in this study. For experiments, HCAECs were grown to confluence, washed with phosphate buffer (PBS), and kept in serum-free medium for 24 h to synchronize the cells, bringing them all to one phase of cell cycle. Afterward, cells were stimulated with medium containing 20% serum collected from Ao or CS of ACS or SA patients for 12 h and then total RNA was isolated following standard procedure and stored at −80°C for further analysis. All experiments have been performed in triplicates.