Fig 2: Affected CPM contains less nuclear aggregates, low level of PABPN1 and signs of regeneration. a Comparison of the percentage of myonuclei containing aggregates in two muscles from the same patient. VLM vastus lateralis muscle, TAM tibialis anterior muscle, DTM deltoid muscle, SCM sternocleidomastoid muscle, CPM cricopharyngeal muscle. Paired t test ns non-significant; **p < 0,005. b Muscle fibers from affected CPM are smaller than those from SCM and contain less aggregrates. PABPN1: green; dystrophin: red; dapi: blue. Scale bar = 50 µm. c Western-blot analysis of the level of PABPN1 in SCM and CPM from three OPMD patients. As a reference a protein extract from skin (SK) has been loaded. Western-blot is normalized with GAPDH protein levels. The age of the patient is indicated above (y: years). d Embryonic eMyHC immunofluorescence staining on a CPM to demonstrate regenerating fibers. eMyHC: green; laminin: white; DAPI: blue. Scale bar = 50 µm

Fig 3: OPMD xenograft model demonstrate that nuclear PABPN1 aggregates appear soon after regeneration but at a lower amount. a EDL and TA from immunodeficient mice have been removed and replaced by small muscle fascicles taken from biopsy samples (SCM) of OPMD patient. Four months later, regenerated muscle can be collected and frozen for analysis. b Regenerated human muscle tissue (derived from the OPMD patient muscle biopsies) can be differentiated from mouse using human-specific labeling of muscle fibers (hspectrin) and muscle nuclei (hlamAC). c H&E and Gomori trichrome staining on the initial biopsy and on the xenografted SCM muscle highlighted features of dystrophic muscle such as centronucleated fibers and rimmed vacuoles. Scale bar = 50 µm. d Immunofluorescence displays nuclear aggregates on an OPMD SCM muscle biopsy prior to xenotransplantation (left) and after xenotransplantation (right). e Percentage of human nuclei containing aggregates before and after xenografting of three independents human OPMD SCM muscle biopsies. f A large nuclear aggregate containing HSP70 in the xenografted SCM muscle four months after engraftment. Scale bar = 10 µm

Fig 4: The mean percentage of nuclei containing aggregates does not vary with PABPN1 genotype but varies with age. a PABPN1 staining (green) performed after a KCl 1 M pre-treatment on 5 μm-thick cryosections. Nuclei are counterstained with dapi (blue) and muscle fibers delineated with an anti-dystrophin (red) staining. Scale bar = 50 µm. Aggregates are indicated with an arrow. b Box-plot and individual values of the mean percentage of myonuclei containing aggregates for each genotype. Within one genotype, the age of the patients is variable. Each dot represents a patient. Mean and SEM are indicated for each genotype. Muscle biopsy samples used: 10–11: SCM, 10–12: SCM, 10–13: SCM/DTM, 10–14: SCM-DTM, 10–15: SCM/DTM/TAM/VLM, 10–16: VLM/TAM, 10–17: VLM, 11–11: SCM/DTM, 13–13: SCM. No statistical difference is observed using a one-way Anova test on all the genotypes except (GCN)10–11, (GCN)10–17, (GCN)11–11 and (GCN)13–13 containing only one or two patient. c Correlation between age at biopsy and percentage of nuclei with aggregates for the 41 patients genotyped (GCN)10–13. Each dot represents a patient. Error bar represents experimental error on the measure. Linear regression statistic test: slope: 0.1401 ± 0.03587; R2 = 0.1080; p < 0.0002

Fig 5: The composition of nuclear aggregates is heterogeneous. a HSP70 and PRMT1 co-staining in PABPN1-aggregates. b Percentage of nuclear aggregates containing HSP70 varies with the genotype. Muscle biopsies used: 10–12: SCM, 10–13: SCM/DTM, 10–14: SCM-DTM, 10–15: SCM/DTM/TAM/VLM, 10–16: VLM/TAM, 10–17: VLM. One-way Anova and post-test for linear trend indicates that linear trend is significant p < 0.001