(CTG)n repeat-mediated dysregulation of MBNL1 and MBNL2 expression during myogenesis in DM1 occurs already at the myoblast stage

Fig 1. RNA expression of MBNL isoforms in myogenic cell cultures with and without expanded (CTG)2600 repeat. (A) Schematic outline of the CRISPR/Cas9 editing procedure by which the (CTG)2600 repeat was excised from the DMPK gene in the DM1 myoblast cell line named DM11 [23]. As a result, a panel of eight isogenic myoblast cell lines was obtained: four non-edited clonal cell lines with expanded (CTG)2600 repeat (the parental cell line and three independent clones) and four clonal lines from which the mutant repeat was removed. (B) MBNL1, MBNL2 and MBNL3 expression in proliferating myoblasts determined by RNA-seq. (C-E) Quantification of MBNL1, MBNL2 and MBNL3 RNA levels in proliferating myoblasts (day -2) and day 0 or 5 of myogenic differentiation measured by RT-qPCR. All bars show the mean values for the four cell lines with and without (CTG)2600 repeat expansion, all values are relative to the parental cell line.

Fig 2. Alternative splicing of MBNL1 and MBNL2 during myogenic differentiation in vitro in presence and absence of an expanded (CTG)2600 repeat. (A, B) Gene structures of MBNL1 and MBNL2, adapted from [1,11]. Boxes represent exons, numbered 1–10, connected by horizontal lines, the introns. Exon lengths are indicated in base pairs. Primers for RT-PCR are shown as red arrows. (C, D) The main alternative splice modes for MBNL1 and MBNL2 identified in this study and the molecular weights of the corresponding protein products, adapted from [1,11]. (E, F) Summary of RNA-seq data of the most prominent alternative splice modes of MBNL1 and MBNL2 in proliferating myoblasts with and without (CTG)2600 repeat (averaged data of four cell lines for each condition). Numerals indicate the percentage of reads spanning an upstream exon to a downstream exon. (G, H) Representative QIAxcel images of RT-PCR analyses of the exon 4 to exon 10/9 region in MBNL1 and MBNL2, respectively, in proliferating myoblasts (-2) and at day 0 and 5 of myogenic differentiation. (I, J) Quantification of the RT-PCR data shown in (G, H).

Fig 3. MBNL1 and MBNL2 expression in proliferating and differentiating myogenic cells with and without (CTG)2600 repeat. (A,B) Representative western blots for MBNL1 and MBNL2 from cell lines with (4F9-13/2600) and without (4A3-ΔΔ) the (CTG)2600 repeat. (C,D) Quantification of MBNL1 and MBNL2 expression, including specific variants, in arbitrary units (a.u.) after normalization to β-tubulin expression. Bars summarize data from two cell lines with and two cell lines without (CTG)2600 repeat. Each cell line was measured three times, in independent experiments.

Fig 4. Subcellular localization of MBNL1 and MBNL2 in myoblasts with and without (CTG)2600 repeat. (A, B) Immunofluorescent localization of MBNL1 and MBNL2 in proliferating myoblasts with (4F9) and without (4A3) the (CTG)2600 repeat expansion. Merged images show DAPI in blue, MBNL1 in green and MBNL2 in magenta. Scale bars are 50 μm. (C-F) Quantification of MBNL1 and MBNL2 levels in the nucleus and the cytoplasm in all eight cell lines. Each symbol represents the average intensity in one myoblast.

Fig 5. Subcellular distribution of MBNL1 and MBNL2 in five-day differentiated myotubes with and without (CTG)2600 repeat. (A-D) Immunofluorescent localization of MBNL1 and MBNL2 in myotubes with (4F9) and without (4A3) the (CTG)2600 repeat expansion, after five days in differentiation medium. Images show DAPI in blue, MBNL1 in green and MBNL2 in magenta. Bars are 50 μm in large images and 10 μm in insets. (E-H) Quantification of MBNL1 and MBNL2 levels in the nucleus and the cytoplasm in all eight cell lines. Each symbol represents the average intensity in one multinucleated myotube.