Fig 1: Distribution of MBNL1 and MBNL2 in FECD and control corneal endothelial cells and in corneal tissue. Immunoblotting images of cytoplasmic (C) and nuclear (N) fractions in (A) Lysates from FECD patient-derived corneal endothelial cell line F35T and control donor-derived corneal endothelial cell line HCN19. (B) Lysates from control corneal endothelial tissues. Two independent replicate experiments are shown. Cytoplasmic and nuclear lysates from equal amounts of cells were loaded in each lane for each protein. GAPDH was used as a cytoplasmic marker. Histone was used as a nuclear marker. Estimation by densitometry measurements showed 3% or less MBNLs proteins to be present in nuclei in control endothelial tissues. (C) Immunofluorescence images of MBNL1 and MBNL2 in control corneal endothelial tissue. Nuclear DAPI staining (blue) defines cell nuclei. Scale bars: 10 μm.
Fig 2: Colocalization of MBNL proteins with CUG repeat RNA foci in FECD corneal endothelial cells detected by RNA FISH and immunofluorescence staining. MBNL1 and MBNL2 colocalize with CUG repeat RNA nuclear foci in (A) F35T patient-derived corneal endothelial cell line (22/1500 CTG18.1 alleles) and (B) corneal endothelial tissue from an FECD patient with the TCF4 expansion (27/51 CTG18.1 alleles). Percentage of RNA foci that colocalize with MBNL proteins is analyzed in (C) F35T patient-derived corneal endothelial cells (n = 2 independent experiments) and (D) endothelial tissues from FECD patients with TCF4 expansion (n = 5 FECD tissues). Results are shown as the mean ± SD. At least 50 cells were analyzed for each sample. Scale bars: 10 μm.
Fig 3: Estimating the quantitative relationship between cellular MBNL protein and the capacity for protein recognition by expanded RNA within intronic TCF4 foci. There are approximately 2 million copies of MBNL1 and MBNL2 proteins in patient-derived cultured endothelial cells but only 65,000 copies per cells in corneal endothelium taken from human donor corneal tissue. In human endothelial tissue, most MNBL1 and MBNL2 protein are in the cytoplasm, with only a fraction (estimated at 1950 copies per cell) remaining in cell nuclei. Depending on the number of repeats and the number of foci per cell, expanded CAG repeat foci have the capacity to bind 25-250 MBNL1 and MBNL2 proteins.
Fig 4: Measurement of the copy number of MBNL1 and MBNL2 mRNA. Standard curves for qPCR efficiency of (A) MBNL1 and (B) MBNL2 were constructed with a serial dilution of purified standard RNAs. Ct values in each dilution were measured in triplicate. The copy numbers of MBNL1 and MBNL2 transcripts per cell in (C) F35T patient-derived corneal endothelial cells (n = 2 independent experiments), (D) control corneal endothelial tissues (n = 3), and (E) patient-derived expanded TCF4 repeat FECD corneal endothelial tissues (n = 3). Results are shown as the mean ± SD.
Fig 5: Depletion of both MBNL1 and MBNL2 reduces RNA foci in F35T patient-derived corneal endothelial cells. (A) FISH images of CUG repeat RNA foci in F35T patient-derived corneal endothelial cells transfected with the indicated siRNA oligonucleotides. (B) Immunoblotting images of lysates from F35T cells showing the efficiency of siRNA-mediated depletion of MBNL1 or MNBL2 protein by siRNAs complementary to MNBL1 or MBNL2 mRNA. GAPDH was used as a loading control (C). Percentage of cells containing foci and number of foci per 100 cells are shown. Results are shown as the mean ± SD, n = 2 independent experiments. P-value was obtained by t-tests analysis of CM compared with siMBNL1+ siMBNL2. At least 100 cells were analyzed for each sample. CM, scrambled control siRNA; MBNL1mm and MBNL2mm, mismatched control siRNAs based on the sequence of the active siRNAs. Scale bars: 10 μm.
Supplier Page from Abcam for Recombinant Human MBNL2 protein