F6886

Adipocytes were incubated in Krebs Ringer Bicarbonate (KRB) buffer supplemented or not with basolateral medium from Caco-2 cells treated with either predetermined concentrations of the extracts, at a dilution of 1/8, or the pro-lipolytic reference products (Forskolin and Isoproterenol), at 37 °C for 2 h. Forskolin (1 µm, F6886-Sigma) and isoproterenol (0.1 µm, I6504-Sigma) were used as activators of lipolysis. Cell culture media were then collected and frozen at − 20 °C. The lipolytic activity of human adipocytes was assessed by the measurement of glycerol (Glycerol assay, GY105, Randox), and non-esterified fatty acid release (NEFA-HR2 R1 Set, 434-91795 et NEFA-HR2 R2 Set, 436-91995, Wako) in the adipocyte culture medium. Adipocytes were collected for DNA quantification (Quant-iT™ PicoGreen™ dsDNA Assay Kit, P7589, Invitrogen) to normalize glycerol and NEFA concentrations.

Primary hepatocytes were prepared from anesthetized mice by hepatic perfusion with type IV collagenase (Sigma, C5138, 120 U/mL) as described [18 (link)] and analyzed within 24 h of harvest. Primary myoblasts were harvested from neonatal mice by collagenase digestion and pre-plating, as described [19 (link)] and passaged up to 3 times. Cells were treated with glucagon (100 nM) or FSK/ IBMX (forskolin 10 μM/ isobutylmethylxanthine 18 μM; Sigma F6886, I5879) in triplicate and processed for luciferase assays using approximately 15 μg total cell lysate as described [18 (link)]. Luminescence (arbitrary units) was normalized to total protein, expressed as A.U./μg protein or fold change from un-stimulated cells.

RSCs were generated and cultured as described previously55 (link)56 (link). Briefly, passage 9 RSCs were grown on PDL to 80% confluence when they were infected with control GFP or YAP S127A Flag adenovirus (2 μl per 962 mm²) for 16 h in growth medium DMEM/F12 (D6421, Sigma) supplemented with 2 μm forskolin (F6886, Sigma) and 4 μg ml⁻¹ bovine pituitary extract (BT215, Alfa Aesar). For differentiation, RSCs were growth-arrested in defined medium56 (link) for 8 h and then supplemented with 1 mM dbcAMP (D0627, Sigma). Forty-eight hours later cells were scraped and protein extracted by sonication in standard RIPA buffer and processed for western blot.

According to a method^{48 (link)} with some modifications, mouse SCs from postnatal day 1–3 (P1–3) mice were established. Sciatic nerves were isolated, cut into small pieces, and dissociated with 2.5 mg/mL dispase II (D4693, Sigma) and 0.5 mg/mL collagenase type IV (C5138, Sigma) for 30 min. The cells were resuspended in 10 mL basic growth medium containing Dulbecco’s modified essential medium (DMEM), 10% inactivated horse serum (HS; 26050070, Gibco), 4 mM L-glutamine (25030–081, Sigma), 1% penicillin–streptomycin (15140-122, Invitrogen), 2 ng/mL human heregulin-beta 1 (396-HB-050, R&D) and 0.5 μM forskolin (F6886, Sigma), plated in 100 mm Petri dishes pre-coated with 100 μg/mL poly-L-lysine (P1274, Sigma) and 10 ng/mL laminin (L2020, Sigma) and maintained at 37 °C in a 5% CO₂ humidified incubator. Medium was refreshed every two days. According to a method^{49 (link)} with some modifications, mouse SCs from adult mice were established. Sciatic nerves were isolated from 15-week-old mice, cut into 10 mm segments and explanted into 30 mm dishes containing 750 μL high-glucose DMEM with 10% heat-inactivated FBS to facilitate myelin removal and cell recovery^{50 (link)}. Medium was refreshed every two days. After 10 days, nerve explants were dissociated with 2.5 mg/mL dispase II and 0.5 mg/mL collagenase type IV for 30 min. Subsequent steps were identical to those described for postnatal SCs.

We randomly put eligible sheep COCs into the TCM-199 (Gibco) culture medium with or without Forskolin (100 μM) (F6886, Sigma) and IBMX (500 μM) (I5879, Sigma)—10 COCs in each group—and placed them into an in vitro culture in a 5% CO₂ incubator at 39 °C for 0 min, 10 min, and 1 h. The cultured sheep COCs were collected into a 1.5 mL centrifuge tube, to which 150 μL of 0.1 M HCl was added for lysis, and the sample supernatant was collected. An aliquot of each sample was assayed for cAMP levels using a cAMP ELISA kit (581001, Cayman, UK) according to the manufacturer’s protocol. A multifunctional enzyme plate analyzer (CYT-1000, GE HealThCare, Chicago, IL, USA) was used to detect OD values at 412 nm, draw the standard curve, and calculate the cAMP level in samples.

Serum-starved cells were stimulated with human/rat CRH (H-2435, Bachem), forskolin (F6886, Sigma), 8-CPT-cAMP (F1221, Sigma), PDGF (01–305; Millipore) or fetal bovine serum (FBS, Natocor) at the concentrations and time points indicated. After incubations, cells were washed with ice-cold PBS and maintained in ice. When calcium chelator BAPTA-AM (B6769, Life Technologies), antagonists or pharmacological inhibitors were used, cells were pre-treated with the drugs or vehicle 15–30 min before stimulation. CRHR1-specific antagonist DMP696 was a generous gift from Dr. Hausch (Max Planck Institute of Psychiatry, Munich, Germany). The following inhibitors were used: H89 (PKA; 371963 Calbiochem), RpcAMPS (PKA; 1337, Tocris), 2′,5′-dideoxyadenosine (tmACs; 288104, Calbiochem), KH7 (sAC; 3834, Tocris), 2-hydroxyestradiol (sAC; 13019, Cayman), U0126 (MEK1/2; 662005, Calbiochem), PD98059 (MEK1/2; 1213, Tocris). For Western blot assays cells were serum-starved for 6 h in OptiMEM before drug pre-treatments or stimulation.

SCs were cultured and purified as previously described (Gu et al. 2014 (link)). Sciatic nerves were harvested from neonatal rats (1–2 days), cut into small pieces and enzymatically treated with 1% collagenase (17018-029, Gibco, Carlsbad, CA, USA) and 0.125% trypsin (25200-056, Gibco). The cell mixture was then resuspended in DMEM (11965-092, Gibco), 10% FBS (12483-020, Gibco), and penicillin–streptomycin (PS; 15140122, Thermo Fisher Scientific, Cleveland, OH, USA) using a 400-mesh cell screen to make a single-cell suspension, seeded in 50 μg/ml poly-d-lysine (PDL; P-7890, Sigma, St Louis, MO, USA) coated dishes with 10 mM cytosine arabinoside (C6645, Sigma) and 10% FBS in DMEM for 1 day to remove fibroblasts. Afterwards, the medium was changed to supplemented with 5 μM forskolin (F6886, Sigma), 2 ng/ml Neuregulin 1 (Nrg1; 396-HB-050, R&D, MN, USA) and 10% FBS in DMEM. After 48 h, cells were incubated with anti-Thy1 antibody (1:1000, M7898, Sigma) to remove remaining contaminating fibroblasts. SC purity was assessed by S100β (1:500, S2532, Sigma) immunostaining. Passage 2 SCs with > 95% purity were used for all cell experiments.