Anti ve cadherin

Cells were incubated with 0, 150, 300 and 600 μg/mL Fe₂O₃ for 48 h at 37 °C. HUVECs were lysed in RIPA buffer and analyzed by western blot. Primary antibodies were as follows: anti-VE-cadherin (#2500S, Cell Signaling Technology, Danvers, MA), anti-CD31 (#3528, CST), anti-α-smooth muscle actin (#19245S, CST), anti-FSP (#13018, CST) and anti-GAPDH (sc-25778, Santa Cruz). Protein bands were quantified using ImageJ software. For the rescue experiment, HUVECs were co-cultured by Fe₂O₃ with 0.88 mg/mL mannitol or 8.8 mg/mL ascorbic acid (AA).

Erucin was produced by myrosinase-catalyzed hydrolysis of glucoerucin isolated from Eruca sativa Mill. defatted seed meals according to Citi et al. [21 (link)]. Erucin was dissolved in dimethyl sulfoxide (DMSO; Merck KGaA, Darmstadt, Germany) and this solution (10⁻² M) was freshly diluted in the appropriate culture medium.
An aqueous solution of d-(+)-Glucose (45% w/w; Merck KGaA, Darmstadt, Germany) was diluted before each experiment in culture medium up to a final concentration of 25 mM. CelLytic™ MT Cell Lysis Reagent, Fluoromount Aqueous Mounting Medium, and 3 kDa FITC-Dextran were obtained from Life Technologies (Carlsbad, CA, USA). Anti-VE-Cadherin was obtained from Cell Signalling (Danvers, MA, USA) and anti-ZO-1 was obtained from Life Technologies (Carlsbad, CA, USA). Anti-SOD-1, anti-iNOS, anti-NF-KB and anti-p22phox antibodies were obtained from Santa Cruz Biotechnology (Dallas, TX, USA). Anti-catalase and anti-β-actin were obtained from Merck KGaA, (Darmstadt, Germany).

Recombinant human TNF-α was purchased from R&D Systems (Wiesbaden-Norderstedt, Germany). Anti-PAR-1 (ATAP2), anti-NQO1, anti-eNOS, and anti-VCAM-1 were from Santa Cruz Biotechnology (Santa Cruz, CA, USA), and anti-phospho-HDAC5, anti-HDAC5, anti-phospho-ERK5, anti-ERK5, anti-phospho-AKT, anti-AKT, anti-phospho-AMPK, anti-AMPK, anti-phospho-eNOS, anti-EEA1, anti-phospho-Src, anti-phospho-FAK, anti-phospho-Erk1/2, anti-Erk1/2 and anti-VE-cadherin were from Cell Signaling Technology (Danvers, MA, USA). Anti-COX-2 was from Cayman Chemical Company (Ann Arvor, MI, USA). Anti-tubulin was from Sigma–Aldrich (St. Louis, MO, USA). Phenylephrine, Acetylcholine and U46619 were purchased from Sigma–Aldrich (St. Louis, MO, USA). Pertussis toxin (PTX) was purchased from Gibco. Small interfering RNA against human PAR-1 was purchased from Santa Cruz Biotechnology (sc-36663, Santa Cruz, CA, USA). For PAR-1 silencing, HUVECs were transiently transfected with 100 pmol/L of control RNA or siRNA targeting human PAR-1 using Lipofectamine 2000 reagent (#11668-019, Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. A non-specific control siRNA from Bioneer was used as a negative control. HUVECs were harvested 48–72 hours after siRNA transfection, protein expressions were assessed by immunoblotting with antibodies and mRNA levels by quantitative real time RT-PCR (RT-qPCR).

HUVEC were grown to confluence in ibidi µ-slide 8 well coverslips (cat# 80826). In experiments to assess cell death, a cell viability 660 stain (eBioscience, cat# 65-0864-18) was used at 1:1000 for 20 min prior to 4% paraformaldehyde fixation (Alfa Aesar, Thermo Fisher Scientific, Heysham, UK). After washing with phosphate-buffered saline (Sigma Aldrich), samples were incubated overnight with anti-VE cadherin (Cell Signaling Technology, MA, USA, cat# 2500, 1:200). The primary antibody was removed, and fluorophore-conjugated anti-rabbit (Thermo Fisher Scientific, cat# 11-4839-81, 1:300) was applied alongside 2.5 µg/ml bisbenzimide (Sigma Aldrich). Fluorescent images were acquired by confocal scanning laser microscopy (Leica SP5). Images were analysed using the free platform FIJI [23] (link), using the ‘Analyse particles’ function to count nuclei.

The following antibodies were used for immunostaining: anti-VE-cadherin XP (Cell Signaling Technologies, Danvers, MA); Rhodamin-phalloidin for direct F-actin staining (Invitrogen Corporation, San Diego, CA) and DAPI (Thermo Fisher Scientific). Alexa 488 and Alexa 555 were the secondary antibodies (Thermo Fisher Scientific, Waltham, MA). For protein analysis: anti-VE-cadherin (Cell Signaling Technologies, Danvers, MA) and anti-β-Tubulin (Cell Signaling Technologies, Danvers, MA). Secondary antibodies were from Invitrogen, Paisly, UK.

The antibodies used in this study were obtained from the following sources: Rabbit anti-ZO-1 (#D7D12) and anti-VE-cadherin (#D87F2) from Cell Signaling Technology (Danvers, MA, USA); Rabbit anti-GLUT-1 (#ab15309) and anti-SGLT-1 (#ab14686) from Abcam (Cambridge, MA, USA); Rabbit anti-GLUT-4 (#sc-7938) from Santa Cruz Biotechnology (Santa Cruz, CA, USA); Donkey anti-rabbit (#NA934) and sheep anti-mouse (#NA931) HRP-linked antibodies from GE Healthcare (Piscataway, NJ, USA); Mouse anti-Claudin 5 (#35-2500), goat anti-mouse (#A11001) and anti-rabbit (#A21428) conjugated to Alexa Fluor® 488 and 555 from Invitrogen (Camarillo, CA, USA). Sterile culture ware was obtained from Fisher Scientific (Pittsburgh, PA, USA), while other reagents and chemicals were purchased from Sigma-Aldrich (St. Louis, MO, USA) or Bio-Rad laboratories (Hercules, CA, USA). Fluorescein isothiocyanate (FITC) and Rhodamine B isothiocyanate (RITC) dextrans were purchased from Sigma-Aldrich, while Cascade Blue®-dextran was obtained from Invitrogen (Eugene, OR, USA).