Cyclic adenosine monophosphate camp

HiPSC-NPCs were plated onto Poly-L-Ornithine/laminin-coated dishes for further differentiation to neurons using neuron differentiation media (NDM) as described by Palm et al. (2015 (link)), consisting of neurobasal media (Gibco) + N2 Supplement (Gibco) + B27 Supplement (Gibco) + BDNF (BDNF; 10 ng/ml; Peprotech) + (GDNF; 10 ng/ml; Peprotech) + TGF-β3 (1 ng/ml; Peprotech) + cyclic adenosine monophosphate (cAMP; 500 μM; Sigma Aldrich). NDM was changed every 2 days for 6 weeks prior to harvesting of neurons.

Strains were maintained in Synthetic Complete medium. This medium contained 1.7 g/L of yeast nitrogen base (YNB) without amino acids and without ammonium sulphate, 1 g/L of sodium glutamate monohydrate, 20 g/L of glucose and the appropriate Kaiser dropout mix supplement (ForMedium, Norfolk, UK). For the pH_c measurements, cultures were pre‐grown and maintained in low fluorescence medium which contained YNB without amino acids, without ammonium sulphate without folic acid without riboflavin (MP biomedicals, Huissen, The Netherlands) instead of the aforementioned standard YNB. All media were buffered at pH 5.0 with 25 mM sodium citrate, unless indicated otherwise. The OD₆₀₀ indicated as a starting point of the experiments were measured in a Lightwave II table spectrophotometer (Isogen life sciences, The Netherlands).
Cyclic adenosine monophosphate (cAMP) (Sigma‐Aldrich; St. Louis, MO, USA) was added at the indicated concentrations from a 200 mM stock in water adjusted to pH 5 with NaOH. 1NM‐PP1 (4‐Amino‐1‐tert‐butyl‐3‐(1ʹ‐naphthylmethyl)pyrazolo[3,4‐d]pyrimidine; Calbiochem, EMD Millipore, Billerica, MA USA) was used from 2 mM stocks in DMSO. Ebselen (2‐phenyl‐1,2‐benzisoselenazol‐3(2H)‐one; AG scientific Inc, San Diego, CA, USA) was added at 10 µM from DMSO stocks at 10 mM. All stocks were stored at −20°C.

For the experiments performed in the United States, fluoranthene (Flthn; purity 97.2%) was purchased from AccuStandard (New Haven, CT, USA), benzo[a]pyrene (B[a]P; purity ≥ 96%), dimethyl sulfoxide (DMSO) and Lucifer yellow from Sigma-Aldrich (St. Louis, MO, USA) and 1-methylanthracene (1-MeA; purity 99.5%) from Crescent Chemical (Islandia, NY, USA). All PAH stock solutions and forskolin (Sigma-Aldrich) and cyclic adenosine monophosphate (cAMP, Sigma-Aldrich) stock solutions were prepared in DMSO.
For the experiments performed in Germany, benzo[a]pyrene (99.9% purity) was purchased at Sigma-Aldrich (Taufkirchen, Germany), fluoranthene (98.6% purity) from AccuStandard (New Haven, CT, USA), and 1-methylanthracene (97.71% purity) from LGC Standards (Wesel, Germany). B[a]P-tetrol I-1 was purchased from Dr. Albrecht Seidel (BIU—Biochemical Institute for Environmental Carcinogens, Großhansdorf, Germany). The DNA adduct studies and cytochrome p450 studies were all conducted in Germany. All other studies were completed at the University of Colorado Anschutz.

For the N2a cells differentiation experiments, 6·10⁴ cells/ml were seeded on the silicon scaffolds, PLGA replicas and tissue-culture plastic disks and cultured for 1 day with DMEM 10% FBS and for 3 days with DMEM without FBS and with 10 μM retinoic acid (RA; Sigma-Aldrich), until fixation for further analysis. As an alternative differentiation method, FBS deprivation with 300 μM cyclic adenosine monophosphate (cAMP; Sigma-Aldrich) for 3 days, after culture for 1 day with DMEM 10% FBS, was used. TCP disks were also seeded for 4 days with DMEM 10% FBS and a change of medium was performed after the first day of culture.
The optimal concentrations of the differentiation media were determined after preliminary experiments, where RA concentrations between 0,1 μΜ and 20 μΜ and cAMP concentrations between 10 and 1 mM were tested.