Forskolin

The drugs used in these experiments were obtained from the following sources:
17β-Estradiol, aldosterone and IBMX were purchased from Sigma-Aldrich (Poole, Dorset, UK); G1, G15, G36, 2,3-bis(4-Hydroxyphenyl)-propionitrile (DPN), 4,4’,4”-(4-propyl-[1H]-pyrazole-1,3,5-triyl)trisphenol (PPT), 7α, 17β-[9-[(4,4,5,5,5-Pentafluoropentyl)sulfinyl]nonyl]estra-1,3,5(10)-triene-3,17-diol (ICI 182,780) and (2)-2-[4-(1,2-Diphenyl-1-butenyl)phenoxy]-N,N-dimethylethanamine citrate (Tamoxifen citrate) were purchased from Tocris Bioscience (Bristol, UK). Forskolin was obtained from Abcam Biochemicals (Cambridge, UK). We thank Professor Jeffrey Arterburn, New Mexico State University, Las Cruces, New Mexico, USA for initial samples of G36.

iPSCs (1.8 × 10⁵ cells) were seeded in a 12-well plate coated with Matrigel (BD Biosciences, San Jose, CA, USA) containing E8 medium. The next day, the medium was replaced with N2B27 medium [50% neurobasal medium (Life Technologies), 50% DMEM F/12 medium (Life Technologies), 5% N2 supplement (Life Technologies), 10% Supplement B-27 minus Vitamin A (Life Technologies), 0.05% β-mercaptoethanol (Life Technologies), 6 μM CHIR99021 (Cayman), and bone morphogenic protein 4 (BMP4, 25 ng/ml)]. The cells were incubated for another 3 days. On day 4, the N2B27 medium was replaced with Stempro-34 SFM (Thermo Fisher Scientific) containing forskolin (2 nM, Abcam, Milton, Cambridge, UK) and vascular endothelial growth factor (VEGF, 100 ng/ml, R&D Systems). The cells were incubated for another 2 days. On day 6, the medium was replaced with Stempro-34 SFM containing Nutrient Supplement and VEGF (50 ng/ml)^{35 (link)}. The cells were incubated for another 2 days with daily medium changes. On day 8, the cells were harvested and analyzed on a BD LSRFortessa™ Cell Analyzer System (BD Biosciences). The data were analyzed using FlowJo software (Version 10). All experiments were performed in triplicate.

All analyses were performed at 36 hpf. Embryos were exposed to the following compounds in fish facility water from 10 hpf to 36 hpf17 (link)45 (link): 10 μM PGE2 (Sigma), 10 μM Indomethacin (Sigma); 0.5 μM Forskolin (Abcam, ab120058), 0.5 μM H89 (Abcam, ab120341), 20 μM Arachidonic acid (Sigma, A9673). DMSO carrier content was at 0.1%. Equivalent amount of DMSO was added to the media as control. For PGG2 (0.1 mg/ml, Cayman, 17010) and PGH2 (0.1 mg/ml, Santa Cruz, sc201266), embryos were injected with these chemicals in DMSO at the bud stage46 .

Rat primary SCs were cultured in DMEM- low glucose (1g/L) (Lonza) supplemented with 3% fetal bovine serum (FBS, Labtech.com) 1 μM forskolin (Abcam), 200nM L-Glutamine (GIBCO), Neuregulin, 100 μg/ml kanamycin and 800 μg/ml gentamycin (GIBCO) on poly-L-lysine coated tissue culture plates and maintained at 37°C and 10% CO₂.
SCs were infected by co-cultivation at a 1:2 ratio with a ΔRafER expressing producer line that had been pre-treated with mitomycin C (Lloyd et al., 1997 (link)). After two to three days, cultures were transferred to selective media containing 400 μg/ml G418 (GIBCO) and the resulting drug resistant colonies pooled and expanded.
For the differentiation assays, NSΔRafER cells were washed and cultured in serum-free SATO defined medium (Mitchell et al., 2003 (link)). Cells were then induced to differentiate by the addition of 1mM dbcAMP (Sigma). To induce their dedifferentiation, ΔRafER was activated by the addition of 100nM hydroxytamoxifen (Sigma) in ethanol (Harrisingh et al., 2004 (link)).

For cAMP and CREB experiments, primary samples or AML cell lines were exposed to small molecules including (±)-SKF-38393 hydrochloride, R(+)-SCH-23390 hydrochloride, Thioridazine (all sourced from Sigma), Forskolin (Abcam), or antibody for 30 minutes in serum-free conditions, followed by cell lysis in HCL 1N. The supernatant containing cAMP was collected and applied to cAMP direct immunoassay kit (Millipore/Calbiochem) as per the manufacturer’s instructions.