Bq788

Co-cultures in modified DPBS, 1.06 mM CaCl₂, were imaged for 2.5 min in streaming mode, 250 ms exposure, to determine the baseline percentage of cells with transients and the number of transients per cell. After baseline imaging, media were replaced with modified DPBS, containing 0 mM CaCl₂ and/or 1 µM thapsigargin, 1 µM BQ788, 1 µM BQ123 (Tocris–BioTechne), 100 µM atropine sulfate (Sigma-Aldrich), or 100 µM atropine sulfate with 1 µM BQ788. To ensure full replacement of the imaging media with the treatment solution, 10 times the volume of the imaging chamber was perfused through the imaging chamber of a MatTek dish using manual syringe-driven delivery through a modified open perfusion insert (model RC-37F; Warner Instruments). The same field of view imaged for the baseline analysis was imaged after a 20-min incubation with the perfused solution. The fold change in percent melanocytes with one or more Ca²⁺ transients was determined by dividing the number of cells with transients during the treatment imaging period by the number of cells with transients during the baseline imaging period. For agonist stimulation, ACh chloride (Tocris) or endothelin-1 acetate salt (Bachem) was perfused into the chamber at 10× excess volume, to ensure complete replacement of imaging media, at the indicated time during the streaming acquisition.

The complete protocol is described in the Online Supplementary Methods and illustrated in Online Supplementary Figure S1. ET_A antagonist (BQ123, A.G. Scientific^®, San Diego, CA, USA or ET_B antagonist (BQ788, Tocris Bioscience^®, Bristol, UK) were injected 10 min before (10 mg/kg) and 3 h 15 min after (5 mg/kg) intrascrotal injection of 0.5 μg tumor necrosis factor (TNFα, R&D Systems, Minneapolis, MN, USA) and neutrophils were monitored by injection of labeled phycoerythrin-conjugated anti-Ly6G antibody (BD Biosciences Pharmingen, Sparks, MD, USA).
The neutrophil rolling flux fraction, adhesion density, adhesion efficiency and transmigration were measured from 2.5 h to 5 h after the TNFα injection at 30-min intervals.

Isolated trophoblasts were seeded in gelatin pre-coated plates and maintained for 2 days in Keratinocyte medium (KCM, Gibco) with penicillin/streptomycin (Gibco), KCM supplements (Gibco) and 10% (v/v) fetal calf serum (FCS, Thermo Scientific) in a humidified incubator at 37°C, 5% CO₂ in air. Prior to treatments, the cells were cultured under low serum conditions (2% (v/v) FCS) for 24 h. Thereafter, the cells were incubated in the absence (control) or presence of 10 nM or 100 nM ET-1 (Sigma Aldrich, St. Louis, MO, USA) for 24 h. The ETR subtype involved in mediating ET-1 effects was determined by pre-incubating trophoblasts with selective ETR antagonists for 2 h prior to the addition of 100 nM ET-1: BQ-123 (Tocris, Bristol, UK, 1.4 and 11.2 nM) for ETRA and BQ-788 (Tocris, 1.2 and 9.6 nM) for ETRB. Because late first trimester (GW 11 + 12) placentas gave highest trophoblast yields, these isolations were used in this set of experiments. Trophoblasts were also treated with TNF-α (Sigma Aldrich, 25 ng/ml) either alone or in combination with 100 nM ET-1, or incubated in the absence or presence of 100 nM ET-1 under three different oxygen tensions (1%, 2.5% and 20% O₂) for 24 h.

NaCl, KCl, MgCl₂, KH₂PO₄,CaCl₂, NaHCO₃, glucose, NaHS, l‐cysteine, phenylephrine, ACh, 5‐HT, l‐NIO (N⁵‐(1‐Iminoethyl)‐L‐ornithine dihydrochloride), Tris‐HCl, sodium deoxycholate, sodium dodecyl sulfate, EDTA, Igepal, protease inhibitor cocktail, BSA, Tween 20, β‐actin, methanol, ammonium acetate, acetic acid, creatine phosphokinase, creatine phosphate DEA‐NO, zinc acetate and, inosine triphosphate and stock solution of cGMP, cAMP and cIMP were supplied by Sigma‐Aldrich, (Milan, Italy). Inosine 3′‐5′‐cyclic monophoshate sodium salt was supplied by (Merck, Germany). Anti‐p‐PDE4A and anti‐p‐PDE5 were supplied by Fabgennix (Frisco, TX, USA). Anti‐PDE4A was supplied by ProteinTech (Manchester, UK). Anti‐PDE5 was supplied by Santa Cruz Biotechnology (Heidelberg, Germany). Anti‐eNOS was supplied by BD Transduction Laboratories (USA). Anti‐β‐actin was supplied by Merck (Germany). ODQ, U46619, FR 139317 and BQ788 were supplied by Tocris (Bristol, UK).