Harmine

Wnt signalling activation was achieved by treatment with either 40 mM LiCl or 50 ng/ml Wnt3a (R&D) for 5 hrs. 40 mM NaCl and 0.1% PBS were employed as control treatments, respectively. The DYRK1A inhibitors Epigallocatechin-3-gallate EGCG (Sigma-Aldrich), INDY (Tocris) and harmine (Tocris) were prepared according to manufacturer’s instructions and administered at previously published doses of 25 μM (EGCG), 1–100 μM (INDY) and 10 μM (harmine) for 5 hr. 0.1% Ethanol or 0.1% DMSO were employed as controls for EGCG and INDY/harmine, respectively.

Harmine (Tocris 5075), LDN-192960 (Sigma-Millipore SML0755), KRIBB11 (Tocris 5480), bortezomib (Selleckchem S1013), carfilzomib (Selleckchem S2853), ixazomib (Selleckchem S2180), and oprozomib (Selleckchem S7049) were dissolved in DMSO at a stock concentration of 10 mM and treatments were carried out as indicated. Curcumin (Sigma-Millipore 08511) was diluted in DMSO at a stock concentration of 5 mM in the dark and prepared fresh prior to each experiment and the excess solution was never stored. Curcumin treatment at a final concentration of 5 µM was always carried out in media containing either 1% bovine serum albumin (BSA) or 10% fetal bovine serum (FBS) to maintain maximum stability and to avoid aggregation [15 (link),19 (link)].

The following compounds were used: Harmine (Tocris, 5075), LY411575 (Sigma, SML0506), and EP (Sigma, E47808). Treatment with Harmine was performed from 3 to 5 dpf with fresh solution added once at 4.5 dpf. Treatment with LY411575 and EP was from 15 to 18 dpf. Fresh solution was added for 12 h (overnight), followed by washing and returning to regular fish water with feeding during the day. DMSO was used as a control.