Fig. 6

Activation of canonical Wnt signaling induces translocation of β-catenin to the nucleus in JARID2 depleted cells. a, b Expression of β-catenin is unchanged with loss of JARID2 as assayed for mRNA by qRT-PCR (a) and protein by western blot assays (b). Error bars are S.E.M. n ≥ 3. n.s. represents not significant statistical difference between the means. c, d Localization β-catenin is altered upon JARID2 depletion. C2C12 cells stably expressing scr or shJarid2 were grown to confluency in high serum (UD) and switched to differentiation conditions for 2 days (D2). shJarid2 cells were treated with either 10 mM NaCl or 10 mM LiCl for 2 days in differentiation conditions as indicated. Both nuclear (c) and cytoplasmic (d) fractions of proteins were extracted and probed for ß-catenin, TBP (loading control for nucleus) and TUBULIN (loading control for cytoplasm). Gels were quantified and normalized to respective loading controls (right side of panel). Relative expression was calculated relative to scr UD sample and plotted as bar graphs. Error bars are S.E.M. n ≥ 3. e–g. Canonical Wnt signaling directly regulates Myod1 expression through binding of β-catenin in the distal regulatory region of the Myod1 promoter. C2C12 cells stably expressing scramble shRNA (scr) or Jarid2 mRNA-specific shRNA(shJarid2) were grown to confluency and switched to differentiation conditions for 2 days (D2). shJarid2 cells were treated with either 10 mM NaCl or 10 mM LiCl for 2 days in differentiation conditions. ChIP assays using antibodies against β-catenin and a nonspecific antibody (IgG) were performed using primers spanning three different regions; distal regulatory region (DRR) (e), proximal regulatory region (PRR) (f), and core enhancer (CE) (g) of the Myod1 promoter. Error bars are S.E.M. ***p value < 0.001. n ≥ 3