Cyclopamine

JAR cells were seeded into 96-well plates at 1000 cells/well and cultured for 24 h. Then, the culture medium was replaced with a fresh complete growth medium plus a final concentration of 5 μM cyclopamine (Tocris, 1623, USA) or 0.5 μg/ml recombinant human Shh (R&D, 1845-SH). Complete growth medium plus an equal volume of alcohol was used as a negative control. The maintenance medium was refreshed every two days with the same formulation as in the previous treatment. At the indicated time points, 10 μl Cell Counting Kit-8 (Dojindo, Japan) solution was added to each well and then incubated for 1 h. The absorbance, indicating cell viability, was measured by SpectraMax M5 (Molecular Devices, USA) according to the manufacturer’s instructions.

Doxycycline (Dox) (Sigma, D9891) was added from 3 days post fertilization (dpf) to 7 dpf at a dose of 10 μg/ml to induce kras expression and at 30 μg/ml to induce Myc expression. SU5402 (Tocris, 3300), SU6668 (tocris 3335), IWR1 (Tocris, 3552), cardionogen 1 (sigma, SML0458), cyclopamine (Tocris, 1623) and GANT61 (Sigma, G9048) were first dissolved in dimethyl sulfoxide (DMSO) as stocks and used for larva exposure from 4 to 7 dpf. The working concentrations used in the experiments were 1 μM SU5402, 1 μM SU6668, 10 μM IWR1, 10 μM cardionogen 1, 10 μM cyclopamine and 1 μM GANT61. All of these small molecular inhibitors have been previously tested and validated in zebrafish models, such as SU540240 (link), SU666841 (link), IWRI25 (link), cardionogene 142 (link), cyclopamine43 (link) and GANT6144 (link). The dosages were selected based on the highest all-survival concentrations and/or our validation in previous experiments45 (link)46 (link).

The following reagents were used from the indicated source: Smo agonist (SAG) and Cyclopamine from Tocris (Bristol, UK). 1,2,3,4-Tetrahydro-6-nitro-2,3-dioxo-benzo[f]quinoxaline-7-sulfonamide (NBQX), D-2-amino-5-phosphovaleric acid (D-APV) from the Molecular, Cellular, and Genomic Neuroscience Research Branch (MCGNRB) of the National Institute of Mental Health (NIMH, Bethesda, MD, USA). 1.2- bis (2-Aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid (BAPTA) and BAPTA-AM from Sigma (St. Louis, MN, USA). TrkB- and TrkC-IgG were purchased from R&D system (Minneapolis, MN, USA). Tetrodotoxin (TTX) was purchased from Abcam (Bristol, UK).

The antibodies were obtained from following sources: mouse anti-SMI-312 (Covance), rabbit anti-phospho-GAP43 (Ser-41; Sigma), rabbit anti-GAPDH, rabbit anti-sonic hedgehog, rabbit anti-phospho-PKAc (thr197), rabbit anti-PKAc (Cell Signaling), mouse anti-patched-1 (H-267, Santa-Cruz), rabbit anti-BMP4 (Abcam), rabbit anti-smoothened (Origene), goat anti-mouse Alexa Fluor 488 and 555 (Life Technologies). Empty construct pCDNA3.1 and EF1αLacZ for the expression of β-galactosidase have been described previously (Kharebava et al., 2008 (link)). Myc and FLAG-tagged human Shh expression construct pCMV6-Entry-myc-Flag-Shh from Origene. Vectors expressing mouse smoothened protein pEGFP-mSmo (Chen et al., 2002 (link)) is from Addgene (Plasmid 25395). For GLI1 transcriptional assays, the pGLI1-Luc Reporter plasmid was used (Signosis). Luciferase assay reagents and β-galactosidase quantification kits were from Promega. Cyclic AMP XP assay kit for the cAMP level quantification was from Cell Signaling Technology. Recombinant murine Shh was from Peprotech. Docosahexaenoic and other polyunsaturated fatty acids, essentially fatty acid-free BSA, SQ22536, and α-tocopherol were obtained from Sigma, and URB597 and cyclopamine were from Tocris; SAG was from Calbiochem.

Cyclopamine (Tocris, 1623, USA), recombinant human Shh (R&D, 1845-SH), chloroquine (Sigma, C6628), GANT61 (Selleck Chemicals, S8075), Lipofectamine 3000 (Thermo Fisher Scientific, L3000015), Gli2 shRNA1 (5′-GUACCAUUACGAGCCUCAUUC-3′), Gli2 shRNA2 (5′-CAACGCCCCCCACCCGUAC-3′), Gli3 shRNA1 (5′-UUGAAGGUUGCACAAAGGC-3′), Gli3 shRNA2 (5′-AAGAGAUUAAACUGACUUU-3′), BECN1 shRNA1 (5′-GGATGACA GTGAACAGTTA-3′), and BECN1 shRNA2 (5′-CCCGTGGAATGGAATGAGA-3′).