Journal: PLoS Genetics

Article Title: Cholesterol Metabolism Is Required for Intracellular Hedgehog Signal Transduction In Vivo

doi: 10.1371/journal.pgen.1002224

Figure Lengend Snippet: In vitro analysis also shows a decreased response to hedgehog protein in mouse embryonic fibroblasts (MEFs). Wild-type, but not mutant, MEFs show increased cell growth upon treatment with SHH (A; *p = 0.001 for wt; n = 4). Quantitative RT-PCR analysis also shows a reduced response of Gli1 (B; *p = 0.002 for wt, p = 0.01 for rud ; n = 3) and Patched (C; *p = 0.001 for wt; n = 3) upon SHH treatment in mutant MEFs. β-galactosidase accumulates in SHH-treated wild-type; Ptc-lacZ MEFs, but not rud;Ptc-lacZ MEFs (D; *p = 0.005 for wt; n = 4). (E) PzP53Med ( Ptc-lacZ;p53 medulloblastoma) cells infected and clonally selected with control plKO.1 lentivirus respond to SHH treatment with accumulation of β-galactosidase (* p = 0.07 for wt; n = 4). Two independent clonal lines infected with Hsd17b7 RNAi constructs do not respond as robustly to SHH treatment.

Article Snippet: Mouse embryonic fibroblasts were generated using standard methods and plated at a density of 20,000 cells/cm 2 in the presence or absence of 200 ng/mL SHH amino terminal peptide (R&D Systems).

Techniques: In Vitro, Mutagenesis, Quantitative RT-PCR, Infection, Control, Construct

Journal: PLoS Genetics

Article Title: Cholesterol Metabolism Is Required for Intracellular Hedgehog Signal Transduction In Vivo

doi: 10.1371/journal.pgen.1002224

Figure Lengend Snippet: Wild-type (A–I) and rudolph (J–R) mutant MEFs were transfected with a SMO-GFP plasmid and acetylated α-tubulin immunocytochemistry was used to identify the primary cilium. SMO-GFP in untreated cells is seen outside the cilium (A–C, J–L), accumulating at the base of the axoneme (not shown), or throughout the cilium (D–F, M–O). Treatment with SHH protein localizes SMO-GFP throughout the cilium in the majority of cells observed (G–I, P–R). All images were taken at the same magnification and the scale bar in C represent 10 µm.

Article Snippet: Mouse embryonic fibroblasts were generated using standard methods and plated at a density of 20,000 cells/cm 2 in the presence or absence of 200 ng/mL SHH amino terminal peptide (R&D Systems).

Techniques: Mutagenesis, Transfection, Plasmid Preparation, Immunocytochemistry