GDC-0449 downregulates the expression of various components of SHH signaling pathway in pancreatic CSCs

<p>. (A), Pancreatic CSCs were treated with GDC-0449 (10 µM) for 36 h. At the end of incubation period, RNA was extracted and the expression of Gli1, Gli2, Patched-1, Patched-2, Smoothened and Shh was measured by qRT-PCR. Data represent the mean ± SD. @ significantly different from respective control (P<0.05). (B), Pancreatic CSCs were treated with and GDC-0449 (0, 1, 5 and 10 µM) for 48 h. Nuclear extracts were prepared and the gelshift experiment was performed. Probe only, control (without GDC-0449), and GDC-0449 treated samples (1, 5 and 10 µM), respectively. The data are representative of three experiments. (C), Gli-dependent luciferase activity is reduced by GDC-0499. Pancreatic CSCs were transduced with lentiviral construct expressing Gli-dependent luciferase reporter, and treated with GDC-0449 (0, 5 and 10 µM) for 48 h. Lysates were prepared, and luciferase activity was measured. Normalized luciferase activity is presented as mean ± SD. @ or # significantly different from respective control (P< 0.05). (D), GDC-0449 inhibits expression of Gli1 and Gli2 in human pancreatic CSCs. The cells were seeded on fibronectin-coated coverslips and treated with GDC-0449 (10 µM) for 48 h. Subsequently, cells were fixed with 4% paraformaldehyde, blocked in 10% normal goat serum and stained with Gli1 and Gli2 primary antibodies (1∶100) for 16 h at 4°C and washed with PBS. Afterwards, cells were incubated with fluorescently labeled secondary antibody (1∶200) along with DAPI (1 mg/ml) for 1 h at room temperature and cells were mounted and visualized under a fluorescent microscope. For better visuality, the color of DAPI was changed from blue to red.