Gsk 3β inhibitor sb216763

Neonatal rat ventricular cardiomyocytes (CMs) were subjected to hypoxia on the fourth day after siRNA transfections. For normoxia cells, DMEM (11966-025, Gibco, Dublin, Ireland) supplemented with 10 mM glucose was used. For hypoxia cells, DMEM supplemented with 10 mM deoxyglucose and 1 mM sodium dithionite (Fluka, Buchs, Switzerland) was used. During hypoxia, cells were incubated in C-Chamber (BioSpherix, New York, NY, USA), with oxygen level controlled at 0.1% with ProOx C21 (BioPherix, New York, NY, USA). When needed 3 µM GSK-3β inhibitor SB216763 (1616, Tocris Bioscience, Bristol, United Kingdom) was added to CMs 20 min before hypoxia experiment.

5aza and PKH2 green fluorescent cell linker kit were obtained from Sigma-Aldrich (St. Louis, MO, USA). Lipofectamine 2000, and SYBR GreenER were from Invitrogen (Grand Island, NY, USA). The MMLV reverse transcription system and dual luciferase reporter assay system were from Promega (Madison, MI, USA). TaqMan MicroRNA Assay, TaqMan MicroRNA Reverse Transcription kit, and TaqMan Universal PCR Master Mix were from Applied Biosystems (Foster, CA, USA). Antibodies, including total and phospho-GSK-3β (Ser 9), total and phospho-Akt (Ser 473), were from Cell Signaling Technology (Beverly, MA, USA). Biotinylated Sca-1 antibody was from BD Biosciences (San Jose, CA, USA). Antibodies for GAPDH and Arrb2 were from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The cardiac troponin T (cTnT) antibody was from Abcam (Cambridge, UK). GSK-3β inhibitor SB216763 was from Tocris Bioscience (Bristol, UK).