Fig 5. CGG repeat expansions in the FMR1 gene inhibit translation of Venus-ARC RNA in human fibroblasts.

Panel A—Human fibroblasts from control individuals C0603 (control male, 31 repeats), GM00497 (control male, unknown repeat number), GM00498 (control male, unknown repeat number) and FMR1 premutation carriers FX08-2 (female, 31, 105 repeats), FX11-2 (female, 20, 79 repeats), FX13-2 (female, 33, 85 repeats), and WC26 (female, two premutation alleles, 60, 90 repeats) were microinjected with Venus-ARC RNA and after 2 hours Venus-ARC RNA and newly-synthesized Venus-ARC protein were imaged by dual channel confocal microscopy. Representative images are shown for untreated control and premutation cells and for premutation cells treated with TMPyP4. Scale bars indicate 5 micrometers. Panel B—Cumulative Kolmogorov Smirnov plots for specific translational activities (newly-synthesized Venus-ARC protein/microinjected Venus-ARC RNA) in individual cells for 3 control and 4 premutation fibroblast cell lines as described for Panel A, untreated (top) and treated with TMPyP4 (bottom). Panel C—Frequency distribution plots for specific translational activities (newly-synthesized Venus-ARC protein/microinjected Venus-ARC RNA) in cells for 3 control and 4 premutation fibroblast cell lines as described for Panel B, untreated (top) and treated with TMPyP4 (bottom). Panel D—PCR analysis of CGG repeat numbers in FX08-02, FX11-02, FX13-02 and C0603 fibroblasts with a 100 bp DNA ladder (left panel) and in WC26 and C0603 fibroblasts with a 1 kb ladder (right panel). In the panel on the left, in the FX08-2, FX11-2, and FX13-2 lanes, the band at the bottom of the gel represents female gender specific PCR product, the bands immediately above the gender specific band represent PCR products from CGG repeat alleles in the normal range and the bands above represent PCR products from expanded CGG repeat alleles. The fainter products near the top of the gel are of unknown origin. In the C0603 lane, the two bands near the bottom of the gel represent male and female gender specific PCR products, and the band above the gender specific bands represents PCR product from the normal CGG repeat allele. In the panel on right, the two bands in the WC26 lane both represent PCR products from expanded CGG repeat alleles and the single band in the C0603 lane represents PCR product from the CGG repeat allele in the normal range. Panel E—Table showing ID, gender, FMR1 alleles and CGG repeat numbers (based on panel D) for each cell line. Panel F—Western blotting of FMRP expression in full mutation (FX08-1, FX11-01, FX13-01) and premutation (FX08-2, FX11-02 and FX13-02) cell lines with actin loading controls.