Wallac victor spectrophotometer

PDOs were collected and dissociated in cold lysis buffer (Biosource International, USA) supplemented with Phosphatase Inhibitor Cocktail 2 (P5726-Sigma Aldrich, Missouri, USA), Phosphatase Inhibitor Cocktail 3 (P044-Sigma Aldrich, Missouri, USA), Protease Inhibitor Cocktail (P8340-Sigma-Aldrich, Missouri, USA) and PMSF (Sigma-Aldrich, Missouri, USA). Protein concentration was determined using Pierce BCA Protein assay kit (ThermoFisher Scientific, USA) and Wallac Victor spectrophotometer (Perkin Elmer, Massachusetts, USA) and loaded in Criterion TGX Precast gel (BioRad, California, USA). Membranes were incubated overnight at 4 °C with the following primary antibodies: anti-DBH (CSB-PA958833, Flarebio biotech LLC., MD, USA); anti-LGR-5 (CSB-PA267899, Flarebio biotech LLC., MD, USA); anti-MycN (CSB-PA965036, Flarebio biotech LLC., MD, USA); anti-p75 NGF receptor (ab93934, Clone 2F1C2, Abcam, Cambridge, UK); anti-Vimentin (NB100–74564, Clone J144, Novusbiologicals, Colorado, USA); anti-GAPDH (NB300–221, Clone 1D4, Novusbiologicals, Colorado, USA); anti-YAP (#4912, Cell Signaling, Massachusetts, USA). Images were acquired using Alliance 9.7 Western Blot Imaging system (Uvitec limited).

Cyclic GMP cell content was evaluated as marker of NOS and sGC activity. At day 14 mEBs were washed with warm Dulbecco's Modified Eagle's Medium (DMEM) and incubated with 100 μM 3-isobutyl-1-methylxanthine (IBMX), a nonspecific inhibitor of cAMP and cGMP phosphodiesterases (IBMX-DMEM) for 15 min at 37°C. Then, mEBs were treated according to Table 1 in a total volume of 10 mL.
All drugs were dissolved in IBMX-DMEM and incubation was performed at 37°C. At the end of treatments, mEBs were rapidly frozen and maintained at −20°C until use. cGMP (pg/mg of total proteins) was measured by ELISA kit (Enzo Life Science, Exeter UK). Briefly, mEBs were recovered in 200 μL ice-water and sonicated on ice with a wave sonicator for 30 min and then centrifuged (12000 ×g for 10 minutes at 4°C). Twenty μL of samples was used for protein determination (BCA method). Each sample was acidified (200 μL of HCl 0.1 M, after 15 min at room temperature), hard vortexed, and then centrifuged (12000 ×g for 10 minutes at 4°C). cGMP quantity was measured using ELISA kit according to manufacturer's protocol and absorbance was read in a Wallac Victor spectrophotometer (Perkin Elmer, Milan, Italy). IBMX and S-nitroso-N-acetyl-DL-penicillamine (SNAP, S-nitrosothiol which serves as a NO-donor) were purchased from Sigma Aldrich.