After being washed with PBS, cells were incubated with 0.5% Triton X-100 in PBS for 15 minutes at room temperature, then blocked with PBS, containing 5% bovine serum albumin, for 60 minutes at 37°C. Cells were subsequently BI 2536 price incubated with anti-SMO antiserum (H-300, 1:250; Santa Cruz Biotechnology, Santa Cruz, CA) at 4°C overnight. After being washed, coverslips were incubated with Texas Red-X goat antirabbit immunoglobulin G (T6391, 1:1,000; Invitrogen, Carlsbad, CA) for 1 hour in the dark. Cells were then washed three times in PBS,

one time in water, and mounted using Prolong Antifade (Invitrogen). The slides were analyzed by fluorescent confocal microscopy (LSM 510; Carl Zeiss, Jena, Germany). In additional experiments, SMO trafficking was examined by total internal reflection fluorescence (TIRF) microscopy.31 KMCH-1 cells cultured on coverslips were transfected with GFP-SMO

plasmid 48 hours before study. Cells were treated as indicated and NVP-LDE225 order fixed with ddH2O, containing 2.5% formaldehyde, 0.1 M of piperazine-N,N′-bis(2-ethanesulfonic acid), 1.0 mM of ethylene glycol tetraacetic acid, and 3.0 mM of MgSO4 for 20 minutes at 37°C. Cells were then washed three times in PBS, one time in water, and mounted using Prolong Antifade (Invitrogen). Slides were analyzed with a TIRF microscope (AxioObserver.Z1; Carl Zeiss). GFP-SMO localized to the plasma membrane was quantified using image analysis software (AxioVision 4.8.2.0; Carl Zeiss). Data are expressed as the average fluorescence intensity in the cell multiplied by the number of pixels above the background. To determine GLI activity, a reporter containing eight directly repeated copies of a consensus GLI-binding site (8×-GLI) downstream of the luciferase gene was employed (pδ51LucII plasmid; δ-crystalline promoter).32 The 8×-GLI reporter was kindly provided by M. Fernandez-Zapico (Division of Oncology Research, Mayo Clinic, Rochester, MN). The 上海皓元 plasmid

was transfected into normal, stable scrambled, or short-hairpin RNA targeting SMO (shSMO) KMCH-1 cells (0.5 μg/well), using FuGene HD (Roche Diagnosis, Basel, Switzerland). Cells were cotransfected with 50 ng of a plasmid expressing Renilla luciferase under the control of cytomegalovirus (pRL-CMV; Promega, Madison, WI). Twenty-four hours after transfection, cells were treated as indicated, cell lysates were prepared, and both firefly and Renilla luciferase activities were quantified using the Dual-Luciferase Reporter Assay System (Promega), according to the manufacturer’s instructions. Firefly luciferase activity was normalized to Renilla luciferase activity to control for transfection efficiency and cell numbers. Data (firefly/Renilla luciferase activity) are expressed as fold increase over vehicle-treated cells transfected with the 8×-GLI/pRL-CMV reporter constructs. All animal studies were performed in accord with and approved by the institutional animal care and use committee.