Fig 1: TGF-ß increases osteocyte responsiveness to Wnt ligand. mRNA levels of Fzd8, Axin2, Ctnnb1, and Lef1 (A) in OCY454s treated with TGFß for 24 h are determined (n = 3 replicates/group, mean ± SEM). Relative luciferase activity (RLU) of Wnt reporter (TOPFlash/FOPFlash) (B) in OCY454 treated with or without TGFß and Wnt3a (n = 6 replicates/group, mean ± SD). TGFß and Wnt3a regulate the level of active ß-catenin (C and D) and phosphorylated and total GSK3ß (E and F), relative to ß-actin, in OCY454 cells (n = 3 replicates/group, mean ± SD). *p < .05 different from control group, # p < .05 different from Wnt3a group, and $ p < .05 relative to TGFß group, One-way ANOVA followed by Holm-Šídák's post hoc test
Fig 2: miR-100 directly targets multiple components of the Wnt/ß-catenin signaling pathway. The 3' UTR of FZD5 (seed region: 3721–3727), FZD8 (seed region: 508–514), DAAM1 (seed region: 461–473), and SOST (seed region: 409–415) contain putative miR-100 binding sites as shown in (A). Cloning into the psiCHECK-2 reporter showed that these 3' UTR fragments of mouse FZD5, FZD8, and DAAM1, but not SOST, are sufficient to confer reporter sensitivity to miR-100. Normalized luciferase activity of 3' UTR constructs (B) in OCY454 cells transfected with miR-100 Inhibitor (miR-100I) or control inhibitor (CI), are reported (n = 6 replicates/group, mean ± SD). OCY454 cells transfected with miR-100I (red) or CI (black) show miR-100 sensitivity of Fzd5 (C) and Fzd8 (D) mRNA levels (n = 3 replicates/group, data derived from 3 experiments are shown as mean ± SEM). Western blot (E and F) shows 2-fold increase in FZD8 protein levels, normalized to ß-actin, in miR-100I transfected OCY454 cells, relative to CI (n = 3 replicates/group, mean ± SD). The inhibitory effect of miR-100 on Wnt signaling is evident from the increased expression of ß-catenin (Ctnnb1) mRNA (G) and active ß-catenin protein levels (H and I) (n = 3 replicates/group, for G data are shown as mean ± SEM and mean ± SD for I). Relative luciferase activity of TOPFlash/FOPFlash (J) in OCY454 cells co-transfected with miR-100I or CI and treated with vehicle or CHIR for 18–20 h (n = 6 replicates/group, mean ± SD). *p < .05 relative to the CI group and # p < .05 relative to the untreated CI group, One-way ANOVA followed by Holm-Šídák's post hoc test
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