Fig. 5
Parathyroid hormone (PTH) induces TG-interacting factor 1 (Tgif1) expression through activator protein 1 (AP1). a Immunoblot of Tgif1 expression in osteoblasts upon PTH or vehicle (Veh) treatment (representative image of 4 experiments). b Tgif1 expression in differentiating SW3 cells upon PTH or Veh treatment (N = 6, 6). c Tgif1 expression in tibiae of Dmp1-CreTg;PTHR1fl/fl mice compared to Dmp1-Cre−;PTHR1fl/fl control littermates after Veh or PTH injection (Dmp1-Cre−;PTHR1fl/fl + Veh, PTH: N = 12, 6, Dmp1-CreTg;PTHR1fl/fl + Veh, PTH: N = 9, 12). d Tgif1 expression in tibiae of Dmp1-caPTHR1 mice compared to wild-type (WT) littermates (N = 5, 6). e Immunoblot of Tgif1 and pCREB in osteoblasts after stimulation with PTH, Forskolin (FSK) or Veh (representative image of 3 experiments). f Schematic of Tgif1 reporter gene constructs. An AP1 binding site (AP1 WT) was mutated (AP1 mut). g Luciferase activity in osteoblasts transfected with a 2.2 kb Tgif1 reporter construct or an AP1-Luc reporter plasmid as positive control and stimulated with Veh or PTH (2.2 kb Tgif1+Veh, PTH: N = 4, 4, AP1-Luc+Veh, PTH: N = 4, 4) h Luciferase activity in ST2 cells transfected with a 2.2 kb Tgif1 reporter construct or different truncations thereof and stimulated with Veh or PTH (2.2 kb Tgif1+Veh, PTH: N = 6, 6, 1.7 kb Tgif1+Veh, PTH: N = 6, 6, 1.2 kb Tgif1+Veh, PTH: N = 6, 6, 0.5 kb Tgif1+Veh, PTH: N = 6, 6). i Luciferase activity in ST2 cells transfected with a WT 2.2 kb Tgif1 reporter plasmid or with an inactivated AP1 site (AP1-mut) and stimulated with Veh or PTH (2.2 kb Tgif1+Veh, PTH: N = 4, 4, AP1-mut+Veh, PTH: N = 4, 4). a, e Immunoblot for Actin was used as a loading control. Normalized fold expression and molecular weight in kilo Dalton (kDa) are indicated. b-d, g-i *p<0.05, **p<0.01, ***p<0.001 vs. 0 Days Veh b, Veh c, g-i or WT d. b #p<0.05, ###p<0.001 vs. 0 Days PTH. Error bars represent the s.e.m. Two-tailed Student's t-test was used to compare two groups c, d, g–i, and analysis of variance (ANOVA) followed by Newman–Keuls post-hoc analysis was used to compare more than two groups b
