Fig. 5

TM-25659 facilitates TAZ nuclear translocation and its binding with Runx2 to potentiate OCN transcription. a TM-25659 treatment significantly enhanced transcriptional coactivator with PDZ-binding motif (TAZ) nuclear translocation and decreased its phosphorylation while it did not affect the overall abundance of TAZ in ADSCs as determined by protein nuclear-cytoplasmic fraction and Western blot assay. b HEK293T cells were infected with TAZ-overexpressing (OP) lentiviral particles and further treated with TM-25659 (10 μM, 24 h). The cell lysates were immunoprecipitated (IP) with a TAZ antibody or an IgG. Exogenous TAZ protein bound with endogenous Runt-related transcription factor 2 (Runx2) protein was analyzed by Western blot. Representative images from three independent experiments are shown. c ADSCs were treated with osteoinductive medium and TM-25659 (10 μM) for 96 h. The cell lysates were then immunoprecipitated with a TAZ antibody or an IgG. Endogenous TAZ protein bound with Runx2 protein was analyzed by Western blot. Representative images from three independent experiments are shown. d Occupation of TAZ in the osteocalcin (OCN) promoter was determined by ChIP assay in ADSCs under osteogenic medium and TM-25659 for 96 h. Results are shown as fold-enrichment relative to IgG IP controls. e Luciferase activity assay from OCN promoter reporter assay in HEK293T cells cotransfected with indicated constructs and pGL-OCN as well as control phRL-CMV plasmids. f Luciferase activity assay from OCN promoter reporter assay in HEK293T cells cotransfected with increased TAZ plasmid together with pGL-OCN, phRL-CMV plasmids. Data shown here are mean ± SD from three independent experiments; ^#P ˃ 0.05, *P < 0.05, **P < 0.01, by ANOVA and Student’s t test. GAPDH glyeraldehyde-3-phosphate dehydrogenase, NC normal control