Pyr10

Acz, L-Phe, Pyr6 and Pyr10 were purchased from Sigma-Aldrich (St. Louis, MO, USA) whereas Pyr3, 2-APB, NPS-2143, and SKF-96365 were purchased from Tocris Bioscience (Minneapolis, MN, USA). All cell culture media including Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), antibiotics (penicillin and streptomycin), glutamine, and Fura-2/acetoxymethyl ester (AM) were purchased from Invitrogen (Carlsbad, CA, USA). All other chemicals used in this study were analytical grade.

CaG, ACZ, neomycin, Pyr6, and Pyr10 were purchased from Sigma–Aldrich (St. Louis, MO, USA). Dulbecco’s Modified Eagle Medium (DMEM) media, fetal bovine serum (FBS), antibiotics (penicillin and streptomycin), glutamine, and Fura-2-acetoxymethyl ester (Fura-2-AM) were purchased from Invitrogen (Carlsbad, CA, USA). All the chemicals were of analytical grade.

BALB/cJ mice were obtained from the University of Valladolid Animal House (10–12-week-old males). The mice were intraperitoneally injected with the TRPC3 specific inhibitor Pyr10 (1 mg/kg) (Sigma-Aldrich) for 1h and then treated with LPS at a sublethal dose of 5 mg/kg (i.p.). For the analysis of proinflammatory factors, animals were sacrificed by ketamine (100 mg/kg) / xylacine (10 mg/kg) administration and cervical dislocation 3 h after LPS treatment. Blood was collected through cardiac puncture. Livers were collected in RNAlater (Ambion) for further analysis by real-time PCR or in protein lysis buffer for western blot analysis. Serum from animals was used for quantification of TNF-α by specific ELISAs (Thermo) following the manufacturer’s instructions. BALB/cByJ- Lpin1fld/J mice carrying a spontaneous mutation in the Lpin1gene (fatty liver dystrophy, fld) were also used [33 (link)]. All procedures involving animals were carried out under the supervision of the Institutional Committee of Animal Care and Usage of the University of Valladolid (Approval No. 7406000), and are in accordance with the guidelines established by the Spanish Ministry of Agriculture, Food, and Environment, and the European Union.