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Fig. 1. GFP-MBNL1 colocalizes with CUG RNA foci and CAG RNA foci in COSM6 cells. (A) Diagram of repeat-expressing construct. Interrupted 960 CUG or CAG repeats are expressed in a human DMPK minigene containing the genomic segment with exons 11-15 (see Materials and Methods). The DMPK minigene without repeats expresses mRNA identical to the truncated DMPK mRNA, but without repeats in the 3'UTR. (B) CUG and CAG RNA foci detected by FISH. COSM6 cells were transfected with DMPK minigenes containing 960 CTG repeats or 960 CAG repeats. Cy3-labeled (CAG)₅ and (CTG)₅ antisense PNA probes were used to detect CUG and CAG repeats, respectively. Nuclei were counterstained with DAPI. (C) Expression of GFP-MBNL1 (71 kDa), was detected by western blot analysis using an anti-GFP monoclonal antibody. (D) CUG and CAG foci colocalize with GFP-MBNL1. Cells were co-transfected with a GFP-MBNL1 expression plasmid and DMPK minigenes containing 960 CTG, 960 CAG or 0 repeats. (E) CUG and CAG foci are RNase-sensitive and DNase-insensitive. GFP-MBNL1 was coexpressed with DMPK minigenes containing 960 CTG or 960 CAG repeats. (F) CUG and CAG foci are not detectable using PNA sense probes (CTG)₅ and (CAG)₅, consistent with the detection of RNA by FISH rather than plasmid DNA. DMPK minigenes containing 960 CTG or 960 CAG repeats were co-transfected with the GFP-MBNL1 expression plasmid. Bars, 10 µM.

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