Shclnd

To investigate β-catenin involvement in the GIP/LOX pathway, β-catenin expression was knocked down in MC3T3-E1 cells using a β-catenin shRNA construct (Sigma Aldrich, catalogue# SHCLND). Cells were transfected with the shRNA construct plasmid using FuGene6 as previously described (Khosravi et al., 2014 ). Cells were also transfected with an empty plasmid as well as a non-target shRNA construct against GFP as controls. Cells were grown to confluence for 24 h and then changed to medium containing 4 μg/mL puromycin to select for transfected cells. After two days, cells were transfected with the previously described wild-type LOX promoter luciferase reporter construct, as well as the renilla luciferase reporter construct. Cells were then serum starved overnight and treated with GIP for 4 h. The data were first normalized by calculating the ratio of firefly to renilla luciferase activity then presented as a fold change between GIP treated and control cells. Protein was also isolated by direct lysis of cells in protein sample buffer and analyzed by Western blot with a β-catenin (Santa Cruz catalogue #7963) and LOX antibody (Abcam, catalogue #Ab31238) to examine their respective protein levels.