Vismodegib

The small molecule inhibitor 13-197 was provided by Dr. Amarnath Natarajan (University of Nebraska Medical Center). Hedgehog inhibitor vismodegib was purchased from LC laboratories (Woburn, MA) and PLK1 inhibitor BI2536 and chemotherapy topotecan were purchased from Selleckchem Company (Houston TX). These inhibitors were dissolved in DMSO at appropriate concentrations and stored at −20°C.

CD4⁺ T cells were treated with the Smo inhibitors cyclopamine (Alfa Aesar, cat no. J61528; 25 mg) or vismodegib (LC Laboratories, cat no. V-4050) at the concentrations indicated. Stock solutions were prepared in DMSO (Life Technologies) for vismodegib and ethanol for cyclopamine at 10 mM and diluted to working concentration in complete IMDM (Gibco). vismodegib stocks were prepared from fresh vials and used within a week.
CD4⁺ T cells were treated with the selective STAT3 inhibitor STATTIC (Bio-Techne, cat no. 2798) and TGFβ signaling inhibitor SB-505124 (Sigma, cat no. S4696) during Th17 polarization. In order to block extracellular Ihh ligand the monoclonal Hh blocking antibody (clone: 5E1, 2BScientific, cat no. Ab01175) or mouse IgG control antibody (Biolegend, cat no. 400370) was used. To block TGFβ signaling, 10 μg/ml anti-TGFβ blocking antibody (clone: 1D11, R&D Systems, cat no. MAB1835) was used.

The HH inhibitor Vismodegib and PI3K-mTOR dual inhibitor BEZ235 were purchased from LC laboratories (Woburn, MA) and a chemotherapeutic drug, cisplatin was purchased from Selleckchem Company (Houston TX). These inhibitors were dissolved in DMSO at appropriate concentrations and stored at −20°C.

All animal care and experimental procedures followed U.S. National Institutes of Health guidelines and were approved by the Animal Care and Use Committee of the National Institute on Aging. Male C57BL/6J mice (Jackson Laboratory, Bar Harbor, ME) were administered by daily oral gavage of 30 mg/kg vismodegib (LC Laboratories, Woburn, MA) in 0.5% methylcellulose and 0.2% Tween 80 (MCT) 34 (link). Animals of both vehicle and vismodegib groups were euthanized after 15 weeks of treatment using isoflurane overdose and tongues were collected from each animal. The length of the study was to allow for at least four taste cell turnovers. Tongues were fixed in 10% neutral-buffered formalin (Sigma-Aldrich, St Louis, MO) for 1 h and then cryoprotected with 20% sucrose in 0.1 mol/L phosphate buffer overnight at 4°C. Serial sections (8–10 μm thickness) were cut through circumvallate papillae using a cryostat (HM 500M, MICRON, GmbH, Germany). In order to obtain a systematic appreciation without bias of the entire papillae, each papilla was sectioned and every 10th section was saved onto a slide. As taste buds are approximately 80–100 μm in length, sampling every 10th section ensured that no two sections were from the same taste bud.