Figure 5

DUX4c gain and loss of function affect cell proliferation. a Representative fields of healthy myoblasts transfected with the indicated pCIneo expression vectors, fixed 24 h later, and processed for immunofluorescence detection of DUX4c (red) and KI67 (green). Arrows point to KI67-positive cells. b The percentage of perinucleolar and nucleoplasmic KI67-positive nuclei on total nuclei was counted in myoblasts showed in a. and represented as the mean ± SD. Either 10 or 15 fields were counted for myoblasts transfected either with pCIneo or pCIneo-DUX4c, respectively (total number of nuclei: 685 and 433, respectively). In addition, the perinucleolar and nucleoplasmic KI67 intensity were measured in each field. The total perinucleolar or nucleoplasmic KI67 intensity (all fields) was divided by the total number of KI67-positive nuclei (either perinucleolar or nucleoplasmic, respectively), and represented as the mean ± SD. The number of nucleoplasmic KI67-positive nuclei was 35 in the healthy culture and 31 in the FSHD culture. For the significance, a Mann-Whitney test was applied. **p < 0.01 (c). Magnification of DUX4c-expressing cells with partial co-localization of nucleolar KI67. d The number of KI67-positive nuclei was counted in 10 fields of healthy and FSHD myoblasts. The percentage of KI67-positive nuclei is represented as the mean ± SD. ***p < 0.001 was considered significant. e Representative fields of FSHD myoblasts transfected with the indicated siRNAs, then fixed 24 h later, and processed for the immunofluorescence detection of KI67. Nuclei were stained with DAPI. Scale bar: 30 μm