Smoothened agonist sag

Control and Ccrk null MEFs were exposed to varying concentrations of Smoothened agonist (SAG, Cayman Chemical), or to fixed concentrations of SAG for varying time periods, followed by harvesting of RNA, with vehicle (DMSO) serving as a negative control. All experiments were performed in triplicate.
In hysteresis experiments, MEFs were induced with 200 nM SAG for 8 hours. The SAG was then washed out and the Smoothened inhibitor Cyclopamine (10 μM, Calbiochem), was added to counteract any signaling that could result from remaining SAG. In other samples, cells were continually exposed to SAG for up to 72 h. The relative, normalized expression of Gli1 was determined using qPCR.

Human NPCs were derived from human fibroblast iPSC by treatment with small molecules as described before [14 (link),15 (link)]. Human induced pluripotent stem cell derived neural progenitor cells used in this study were generated at the Max Planck Institute for Molecular Biomedicine (Münster, Germany) [15 (link)]. NPCs were cultured on Matrigel-coated 12-well cell-culture plates (Nunc, Rochester, New York, NY, USA) in N2B27 medium supplemented with 3 µM CHIR99021 (Axon Medchem, Groningen, The Netherlands), 0.5 µM Smoothened agonist (SAG) (Cayman Chemical, Ann Arbor, MI, USA), and 150 µM Ascorbic Acid (AA; Merck, Darmstadt, Germany) with a medium change every second day. For plate coating, Matrigel (high concentration, growth factor reduced; Corning, New York, NY, USA) was diluted 1:100 in Knockout Dulbecco modified eagle medium (DMEM, Thermo Fisher Scientific, Waltham, MA, USA) for coating with 500 µL per well for 2 h. Cells were split in a 1:9 to 1:12 ratio every 7 days by single cell digestion with prewarmed accutase (Merck) for 10 min at 37 °C. Cells were diluted 1:10 in DMEM (Merck) prior to centrifugation at 200× g for 5 min. After resuspension in fresh NPC medium (N2B27 supplemented with CHIR, SAG, and AA), cells were plated on Matrigel-coated cell-culture dishes.

The generation and culturing of human NPCs has been previously described (37 (link)). NPCs were grown in N2B27 medium (DMEM-F12/Neurobasal at 50:50 supplemented with 1% penicillin/streptomycin/glutamine, 1% B27 supplement without vitamin A, and 0.5% N2 supplement) containing 3 μM CHIR99021 (Cayman), 150 μM L-ascorbic acid (Sigma-Aldrich), and 0.5 μM Smoothened Agonist (SAG) (Cayman). For differentiation, the medium was replaced with N2B27 containing 1 ng/ml BDNF (Miltenyi Biotec), 0.2 mM L-ascorbic acid, 1 μM retinoic acid (Sigma-Aldrich), 1 ng/ml glial cell line-derived neurotrophic factor (GDNF) (Miltenyi Biotec), and 0.5 μM SAG. On day 8, the medium was changed for inducing neural maturation to N2B27 containing 5 ng/ml activin A (Miltenyi Biotec), 0.1 mM dbcAMP (Sigma-Aldrich), 2 ng/ml BDNF, 0.2 mM L-ascorbic acid, 1 ng/ml TGFβ-3 (Peprotech), and 2 ng/ml GDNF. On day 10, the cells were seeded on a four-well plate for immunofluorescence and 2.5 μM N-[(3,5-Difluorophenyl)acetyl]-L-alanyl-2-phenyl]glycine-1,1-dimethylethyl ester (DAPT) (Sigma-Aldrich) and activin were added to the maturation medium. After 2 d, DAPT and acitivin were removed and the cells were further grown in the maturation medium for neural maturation until day 30.

NIH-3T3 cells were co-transfected with plasmids containing Cdkn1b promoter constructs of various lengths upstream of firefly luciferase (provided by Dr D. Everly, Rosalind Franklin University of Medicine and Science, North Chicago, IL)59 (link) and Renilla luciferase (10:1 dilution), followed by transfection of siRNA against 14-3-3ζ or the scrambled control. To determine hedgehog-dependent transcriptional activity, Shh-light2 cells were treated with the synthetic Smoothened agonist (SAG; Cayman Chemicals, Ann Arbor, MI) or transfected with siRNA against 14-3-3ζ or the scrambled control. Luciferase activity was measured after 24 or 48 h with the Dual-Luciferase Reporter Assay system (Promega, Madison, WI). Transcription factor binding sites were analysed with MotifMap34 (link). ChIP analysis of Gli3 binding to the Cdkn1b promoter was performed with antibodies against Gli3 and the Pierce Magnetic ChIP kit, as per the manufacturer's protocol (Thermo Scientific, Rockford, IL).