Figure 5.

Activation of the Wnt/β-catenin signaling reduces DUX4 expression in FSHD myotubes. (A) RT-PCR of non-FSHD and FSHD-derived cells cultured in myoblast proliferation or differentiation medium in the absence or presence of GSK3β inhibitor (GSKi). Transcription of the Wnt target gene, AXIN2, was monitored as a control for activation of Wnt/β-catenin signaling. (B) Primary FSHD(2349) myoblasts were differentiated in medium containing GSKi or 250 ng/ml recombinant Wnt3A (rWnt3A) or relevant vehicle controls (DMSO for GSKi or 0.1% CHAPS for rhWnt3A). Myotubes were fixed at 48 h to quantify DUX4 protein expression. (C) Immunofluorescent microscopy images of cells prepared in (B). Scale Bar = 50 μm. Myotube index for the image displayed in each condition is displayed in white text in the lower left corner of the panel (MI). (D) Primary FSHD(2349) myoblasts were cultured as in (B) and fixed at 96 h to measure myotube loss. Photos from the 96 h timepoint stained with MHC (green) are shown. The percentage of DUX4(+)/MHC(+) nuclei from a 48 h timepoint of the matching experiment are shown in white text in each quadrant. Scale bar = 200 μm. (E) Myotube index of experiments shown in (D). P < 0.05 by Student's t-test. (F) FSHD2 myotubes were treated with GSKi to confirm that the effect of Wnts on DUX4 protein levels was independent of D4Z4 chromatin structure. DUX4(+) nuclei were quantified. P < 0.05 by Student's t-test. (G) C2C12 myoblasts infected with a lentivirus encoding D4Z4(FFL) were cultured in the presence of absence of GSKi, and Luciferase activity was measured. P < 0.05 by Student's t-test.