Fig. 3
Deletion of TG-interacting factor 1 (Tgif1) does not alter canonical Wnt signaling. a Immunoblot of Tgif1 protein expression in wild-type calvarial osteoblasts after stimulation with recombinant Wnt3a or vehicle (Veh) for 4 h. Immunoblot for Actin was used as a loading control. Normalized fold expression and molecular weight in kilo Dalton (kDa) are indicated (representative image of 4 experiments). b Luciferase assays in wild-type calvarial osteoblasts transfected with a 2.2 kb Tgif1 reporter construct or a Wnt-responsive TopFlash reporter plasmid and stimulated with Veh or Wnt3a for 24 h. c ST2 cells were transfected with a 2.2 kb Tgif1 reporter construct and different truncations thereof, and stimulated for 4 h with Veh or Wnt3a. d ST2 cells were co-transfected with empty vector (Ev) or β-catenin and the 2.2 kb Tgif1 reporter construct or truncated forms thereof. b–d **p<0.01, ***p< 0.001 vs. Veh or Ev control. e Relative β-galactosidase (BAT-GAL) mRNA expression in tibiae of BAT-GALTg;Tgif1+/+ and BAT-GALTg;Tgif1−/− mice (N = 4, 4). f Quantification of the fraction of β-galactosidase-positive bone marrow stromal cells (BMSCs) obtained from BAT-GALTg;Tgif1+/+ and BAT-GALTg;Tgif1−/− mice upon stimulation with parathyroid hormone (PTH) for 4 h (N = 5, 5). ***p< 0.001 vs. BAT-GALTg;Tgif1+/+;Veh, #p< 0.05 vs. BAT-GALTg;Tgif1−/−; Veh, §p<0.05 vs. BAT-GALTg;Tgif1+/+;PTH. g Relative mRNA expression of the Wnt pathway target genes CyclinD1 and h Axin2 in tibiae of 12-week-old male mice of the genotype Tgif1+/+ or Tgif1−/− (N = 8, 6). i Relative activation of the canonical Wnt pathway upon stimulation with increasing concentrations of recombinant Wnt3a in calvarial osteoblasts of the genotypes Tgif1+/+ and Tgif1−/− quantified by a TopFlash reporter gene assay (N = 6). ***p< 0.001 vs. cells of the same genotype without Wnt3a stimulation. Error bars represent the s.e.m. Two-tailed Student's t-test was used to compare two groups b–e, g, h, and analysis of variance (ANOVA) followed by Newman–Keuls post-hoc analysis was used to compare more than two groups f, i
