Hedgehog Signaling Pathway Cell Culture

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).