Microscopy Analysis of Fluorescent BODIPY-Cyclopamine Competition Assay

Human wild type Smo (hSmo) expression vector is a gift from Professor Ke Yu (Fudan University, Shanghai, China). Microscopy analysis of fluorescent BODIPY-cyclopamine competition assay was performed as following. In brief, the HEK293 cells were seeded in 6-well plates and transfected with wild type hSmo expression vector. Two days after transfection, the cells were seeded in 24-well plates and incubated with compounds. After incubation for 10 h, the cells were washed with PBS twice, fixed with 4% paraformaldehyde in dark for 10 min, washed with PBS again and incubated with Triton X-100 solution for 10 min. The cells were then stained with DAPI (Sigma-aldrich) and photographed using a fluorescence microscope.