7 8 dihydroxyflavone hydrate

At 3 dpf dye larvae were separated from unaffected sibling larvae based on phenotype. dye mutants and siblings were treated with either 1 µM Trichostatin A (TSA, Sigma) with or without 100–500 nM ANA-12, 6 µM Scriptaid (Sigma), 10 µM MC1568 (Tocris Biosciences), 10 µM MS275 (Cayman Chemicals), 10 µM 7,8-dihydroxyflavone hydrate (Sigma) or 0.1% dimethyl sulfoxide (DMSO) vehicle control in a final concentration of 0.1% v/v DMSO/embryo medium solution. Optimum drug concentrations for treatments were determined by prior treatment with 0.25–4 µM TSA, 2-10 µM Scriptaid, 5–20 µM MC1568 and 5-20 µM MS275, 2.5–10 µM 7,8-dihydroxyflavone hydrate, and assessment of defects in gross morphology and optokinetic response. For each treatment 12 larvae were transferred to 10 mL drug solution in a 60 × 15 mm petri dish, sealed in a container, and incubated under standard conditions stated above until 5 dpf.

7,8-Dihydroxyflavone hydrate (#D5446; purity > 98%; CAS Number 38183-03-8)) was purchased from Sigma-Aldrich (Darmstadt, Germany); ammonium hydroxide solution 28–30% was purchased from Alfa Aesar (Karlsruhe, Germany); and the phosphatase inhibitors Halt® and Phosstop® from Thermo-Fisher/Piercenet (Bonn, Germany) and Roche Diagnostics GmbH (Mannheim, Germany). All solvents were of analytical grade or higher quality. Acetonitrile was obtained from Carl Roth GmbH (Karlsruhe, Germany), 1-butanol, n-hexane, 2-propanol, methanol, acetone, ammonium acetate and assay buffer compounds: Tris–HCl, MgCl₂, ZnCl₂ and Na₂CO₃ were obtained from Merck (Darmstadt, Germany). Octyl-β-d-glucopyranoside and dithiothreitol were from Sigma-Aldrich (Schnelldorf, Germany). Millipore water was used for all solutions (Schwalbach, Germany).