All reagents were obtained from Sigma Chemical Co. (St. Louis, MO) unless indicated otherwise. Culture media, Dulbecco’s modified ABT-263 datasheet Eagle’s medium, HAM’s F12, fetal bovine serum, MEM nonessential amino acids solution, MEM vitamin solutions, glyceryl monostearate,

chemically defined lipid concentrate, soybean trypsin inhibitor, penicillin/streptomycin, gentamycin, and glutamine and were purchased from Invitrogen (Carlsbad, CA). The PKA inhibitor 14-22 amide, myristolated (PKI) was purchased from Calbiochem (La Jolla, CA). Sorafenib was kindly provided by Bayer Pharmaceuticals (Wayne, NJ). Octreotide was purchased from Polypeptide Group (Strasbourg, France) and RAF265 was purchased from Selleck Chemicals (VWR, Randor, PA). The study was performed in normal wild-type (WT) mice and in Pkd2flox/−:pCxCreERTM mice (S. Somlo, Yale University), an ADPKD mouse model characterized previously.7, 8 The latter model, a conditional knockout mouse model abbreviated as Pkd2cKO, Daporinad in vivo is generated by an inducible defect in PC2 (Pkd2flox/−:pCxCreERTM), targeted through a Cre system, and fused to the ligand-binding domain of a mutated estrogen receptor as described previously.7, 8 The deletion of floxed PC2 alleles is achieved Diflunisal 28

days after birth by exposing the mice to tamoxifen (0.2 mg/g/day) for 5 days. Pkd2cKO mice developed a liver phenotype resembling human ADPKD.7, 8 One week after induction, the animals were treated for 8 weeks with: (1) sorafenib tosylate (Bayer Pharmaceuticals Wayne, NJ) given by gavage at a dose of 20 or 60 mg/kg/day; (2) octreotide (Polypeptide Group, Strasbourg France) at a dose of 100 μg/kg twice per day; (3) sorafenib at a dose of 20 mg/kg/day and octreotide

at a dose of 100 μg/kg; and (4) vehicle (Cremophor, 12.5%; ethanol, 12.5%; water, 75%) for sorafenib or phosphate-buffered saline for octreotide. Because there were no significant differences between the two groups treated with vehicle, these mice were considered a single control group and called “vehicles”. The timing of the treatment was based on our prior experience with this mouse model,7, 8 whereas sorafenib and octreotide doses were derived from prior literature on rodents.10, 16, 17 All experiments were performed according to protocols approved by the Yale University Institutional Animal Care and Use Committee. In this study, we used cultured cholangiocytes isolated from Pkd2cKO mice after induction with tamoxifen and their WT littermates as described.