Vismodegib

Actinomycin D (50-76-0; A1410; Sigma) was diluted into DMSO at 10 mg/mL stock. The stock solution was diluted into ASW to a final concentration of 40 μg/mL. This concentration was reported to block transcription in Halocynthia embryos (Miyaoku et al, 2018 (link)). Flavopiridol (146426-40-6; S1230; Selleck chemicals) was diluted into water to 10 mM stock. The stock solution was diluted into ASW to final concentration 1 and 10 μM. The transcriptional inhibitor-treated embryos were fixed by MEM-PFA (4% PFA, 0.1 M MOPS, 0.5 M NaCl, 1 mM EGTA, 2 mM MgSO₄) after 1 h inhibitor treatment, and used for in situ hybridization.
1-Azakenpaullone (S7193; Selleckchem; Feinberg et al, 2019 (link)), Ruxolitinib (INCB018424; S1378; Selleckchem), Vismodegib (GDC-0449; S1082; Selleckchem), DAPT (208255-80-5; D5942; Millipore Sigma), SB431542 (S1067; Selleckchem; Ohta and Satou, 2013 (link)), U0126 (9903; Cell Signaling Technology; (Hudson et al, 2003 (link))) and Dorsomorphin (1219168-18-9; S7306; Selleckchem; (Ohta and Satou, 2013 (link); Feinberg et al, 2019 (link))) were used to perturb define signaling pathways as described in corresponding references. These treatments were done in a final concentration of 10 μM for 2 or 4 h. The inhibitor-treated embryos were fixed by MEM-PFA after 2 h inhibitor treatment, and used for in situ hybridization.

HEK293T, Shh-Light II, Ptch1^–/– MEFs, SuFu^–/– MEFs, wild-type MEFs and 22Rv1 cells were cultured in DMEM plus 10% FBS. DAOY cells were maintained in Eagle's Minimum Essential Medium (MEM) plus 10% FBS. All media contained l–glutamine and antibiotics. ASZ001 BCC cells were cultured in 154CF medium (Gibco-BRL, Grand Island, NY, USA) plus 2% FBS chelated with Chelex 100 sodium form (Sigma Aldrich), calcium chloride 0.05 mM (Gibco-BRL) and antibiotics. Cerebellar GCPs (from 4-days-old mice) were isolated and cultured as previously described.^{94 (link)} TE354T human BCC cells (ATCC CRL–7762) were cultured in DMEM medium (ATCC 30–2002) plus 10% FBS and antibiotics. Murine MBs were isolated from Ptch1^+/– mice (The Jackson Laboratory, Bar Harbor, ME, USA). Tissues were collected as previously described,^{27 (link)} and immediately prepared cell suspensions were used for short-term cultures to keep Hh-sensitivity in vitro.^{58 (link), 59 (link), 60 (link)} Transient transfections were performed using DreamFect^TM Gold transfection reagent (Oz Biosciences SAS, Marseille, France). Cells were treated with SAG (200 nM, Alexis Biochemicals Farmingdale, NY, USA), Bodipy-Cyclopamine (5 nM, BioVision Inc., San Francisco, CA, USA), Cyclopamine (Calbiochem, Nottingham, UK), Vismodegib (Selleckchem, Munich, Germany), LDE-225 (Selleckchem, Munich, Germany).

Cells were treated 24 h with SMO agonists 200 and 400 nM SAG (566660, Calbiochem) and 3 µM purmorphamine (4551, Tocris bioscience), or antagonist 5 µM vismodegib (S1082, Selleckchem), or kinase inhibitors: 10 µM GF109203X (ALX-270-049, Enzo Life Sciences), 10 µM SB431542 (1614, Tocris bioscience), 20 µM PD98059 (9900, Cell Signaling Technologies), 5 µM SP600125 (270-339-M005, Alexis Biochemicals), 10 µM SB216763 (1616, Tocris bioscience), 200 nM Rapamycin (R0161, LKT Laboratories), 50 µM LY294002 (70920, Cayman Chemical), 5 µM AG1478 (270-036-M001, Alexis Biochemicals), 3 µM Gö6976 (12060S, Cell Signaling Technologies), 10 µM Rottlerin (1610, Tocris bioscience), 20 µM ZIP (2549, Tocris bioscience, kind gift from Prof. Anthony Oro, Stanford University School of Medicine).
To induce cilia in proliferating condition, hTERT-RPE1 cells were treated for 16 h with 50 nM cytochalasin D (250255, MERCK).