Cpt camp

Retinal neurons were dissociated from E14.5 to 15.5 mouse embryos by papain in DPBS (1× Dulbecco’s phosphate-buffered saline [PBS], Corning, NY) following the previously described methods (Kechad et al., 2012 (link)), and neuronal suspension was plated on acid-washed glass coverslips pre-coated with poly-D-lysine (Trevigen, 100 μg/ml) for 1 hr and laminin (Trevigen, 5 μg/ml) overnight at 37°C. Culture medium was made up of half DMEM/F12 medium (Gibco) and half neurobasal medium (Gibco), supplemented with B27 supplement (Life, 0.5×), penicillin-streptomycin (Life, 1×), N-2 supplement (Gibco, 0.5×), N-acetyl-L-cysteine (Sigma, NAC 0.6 mg/ml), cpt-cAMP (Sigma, 100 μM), forskolin (Sigma, 10 μM), and insulin (Sigma, 25 μg/ml). EGF (PeproTech, 50 ng/ml), BDNF (PeproTech, 50 ng/ml), NT-3 (PeproTech, 25 ng/ml), and FGF-basic (PeproTech, 10 ng/ml) were freshly added before using.

Primary Schwann cells were treated with or without 250 μM 8-(4-chlorophenylthio) adenosine-3′,5′-cyclic monophosphate (CPT-cAMP) (Sigma). After 72 h, total RNA from primary Schwann cells was extracted with TRIzol reagent (Invitrogen) according to the manufacturer’s protocol. To examine changes in miRNA expression in cAMP-treated Schwann cells, an miRNA array was carried out according to standard protocols described elsewhere [49 (link)].

α,β-dehydrolovastatin (DHLV) and its derivatives, lovastatin and α,β-dehydrodihydromonacolin K, were produced by the soil-derived fungus Aspergillus sclerotiorum PSU-RSPG178, which was deposited as BCC56851 at BIOTEC Culture collection, National Center for Genetic Engineering and Biotechnology (BIOTEC), Thailand [23 (link)]. Mevastatin, pravastatin, and simvastatin (Cat# M2537, P4498, and S6196) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Dulbecco’s Modified Eagle Medium/Nutrient Mixture F-12 (DMEM-F12; Cat#12400–024), fetal bovine serum (FBS; Cat#10270–106), trypsin-EDTA (Cat#25300–062), penicillin/streptomycin (Cat#15140–122) were purchased from Thermo Fisher Scientifc Inc. (Waltham, MA, USA). Cholera toxin (CT; Cat#10654, Lot#10067A1) was obtained from List Biological Laboratories, Inc. (Campbell, CA, USA). Heat-stable toxin (STa; Product no.4044297, Lot#1000007536) was purchased from Bachem (Torrance, CA, USA). Dorsomophin or Compound C (AMPK inhibitor; Cat#P5499), CPT-cAMP (Cat#C3912), genistein (Cat#C6649), ATP (Cat#A5394), forskolin (Cat#F6886), Na₃VO₄ (Cat#S6508), CFTR_inh-172 (Cat#C2992), IBMX (phosphodiesterase (PDE) inhibitor; Cat#I5879), Ouabain (Cat#O3125) were purchased from Sigma-Aldrich (St. Louis, MO, USA). MK571 (Cat# 70720) was purchased from Cayman Chemical (Michigan, USA). Other chemicals were purchased from Merck Millipore (Burlington, MA, USA).

RBECs were grown on collagen/fibronectin-coated semipermeable filters (0.4 μm pore size, 1.12 cm², Costar Corning Transwell Clear, Sigma). After reaching confluence, the endothelial monolayer was supplied with 550 nM hydrocortisone, 250 μM CPT-cAMP (Sigma) and 17.5 μM RO-201724 (Roche) and placed into the wells of the CellZscope instrument (nanoAnalytics, Münster, Germany) containing astrocyte conditioned medium. Treatments were applied after TEER had reached plateau. TEER was recorded at the indicated time points.