Recombinant heregulinβ 1

SCs were isolated, cultured, and enriched as previously described [20 (link),48 (link)]. Briefly, sciatic nerves were digested and cultured in 0.01% poly-L-lysine hydrobromide (PLL, Sigma-Aldrich, St. Louis, MO, USA) and 5 µg/mL laminin (Sigma-Aldrich) coated dishes with Schwann cell culture medium consisting of MEMα (GlutaMAXTM-I, GIBCO, Waltham, MA, USA) supplemented with 2.5% HEPES (GIBCO), 1% penicillin-streptomycin (P/S, GIBCO), 1% sodium pyruvate (GIBCO), 5% (FCS, LINARIS, Dossenheim, Germany), 10 ng/mL recombinant heregulinβ-1 (PeproTech, London, UK), 0.5% N-2 supplement (GIBCO), 2 µM forskolin (Sigma-Aldrich), 10 ng/mL recombinant FGF basic (PeproTech), and 5 ng/mL PDGFAA (PeproTech). rSC cultures from passage 2 (p2) but not higher than p5 were used for experimentation. For the immunofluorescence staining analysis, 1 × 10⁴ rSCs were seeded per PLL/laminin-coated 8-well (μ-slides, Ibidi, Gräfelfing, Germany) in Schwann cell culture medium and grown until desired confluency. For the proliferation assay, 8 × 10³ rSCs were seeded per coated 8-well.

cAMP [8-(4-Chlorophenylthio) adenosine-3',5'-cyclic monophosphate] and AMP (8- Bromoadenosine-5-O-monophosphate) were purchased from Biolog (US distributor, Axxora LLC, San Diego, CA). Recombinant heregulin-β1 (herein referred to as neuregulin) was purchased from Peprotech (Rocky Hill, NJ). Forskolin, poly-l-lysine, bicarbonate, sodium ascorbate, bafilomycin A1, 2,2’-bipyridyl, deferoxamine and laminin from Engelbreth-Holm-Swarm murine sarcoma basement membrane were purchased from Sigma-Aldrich (St. Louis, MO). Caffeine was bought from Enzo Life Sciences (Farmingdale, NY). SQ 22536, IBMX, ESI-09, CGRP (calcitonin gene-related peptide (rat)) and isoproterenol hydrochloride were purchased from Tocris Bioscience (Bristol, UK). KT5720 and H-89 Dihydrochloride were purchased from Millipore (Billerica, MA). LCD (L-cis-Diltiazem) was purchased from Abcam (Cambridge, UK). Dulbecco’s modified eagles’s medium (DMEM) that contain reduced level of sodium bicarbonate (1.5 g/L) (cat#30–2002) was purchased from American Type Culture Collection (ATCC) (Manassas, VA).