Fig. 5.

DUX4 and PITX1 stabilization in facioscapulohumeral muscular dystrophy (FSHD) myotubes treated with MG132. (A) PITX1 (red) was detected by immunofluorescence with the rabbit anti-PITX1 serum in nuclei of myotubes 4 days after the induction of differentiation (a', b'). Phase contrast microscopy was used to visualize the myotube morphology and position of the nuclei (a, b). b and b' correspond to primary myotubes treated with 25 μM MG132, a proteasome inhibitor, for the last 5 hrs in culture before fixation. Arrows indicate the most stained nuclei and the dotted arrows the intensity gradient of PITX1 staining. (B) DUX4 (green) and PITX1 (red) were detected by immunofluorescence with MAb 9A12 or the rabbit anti-PITX1 serum, respectively, in nuclei of FSHD (aFSHD1 and dFSHD12) primary myotubes 4 days after the induction of differentiation. The myotubes were treated with 25 μM MG132 as in (A). Colocalization of DUX4 and PITX1 staining appears yellow (Merge). DAPI (blue) was used to localize nuclei. Arrows indicate positive nuclei for DUX4 and PITX1 staining and dotted arrows indicate negative nuclei for DUX4 and PITX1 staining. (C) Nuclear proteins extracted from aFSHD3 primary myoblast were analysed by 12% PAGE-SDS followed by Western blotting and immunodetection with MAb 9A12 as described in Materials and Methods. Myotubes were treated with 0 or 25 μM MG132 as indicated 5 hrs before harvest. Nuclear extracts were prepared using the NE-PER kit at the proliferation state (pro) or 4 days after the induction of differentiation (diff 4). Total extracts of TE671 cells transfected with pCIneo-DUX4 were used as a positive control (C⁺). DUX4 proteolysis products observed in the absence of MG132 are shown by red braces. Ponceau red staining of the membrane was used as loading control.