Fig 2: 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 3: 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 4: 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 5: TCF4 pathway associations and alteration of cell cycle control. (A) GSEA results for PCCs between TCF4 and any other gene in the TCGA breast cancer dataset. The left and middle panels show the negatively correlated (FDR <5%) pathways linked to DNA repair. Right panel shows the result of the signature of homologous recombination defects and chemoresistance. (B) Left panels show the cell cycle profiles of control (top) or cisplatin (bottom) CAL-51 cells transduced with control pLKO.1 or an shRNA targeting TCF4 expression. The percentages of cells in the G1, S and G2/M phases are shown. Right panels so western blot results for PARP, pH2AX and TUBA (loading control) in the corresponding cell assays. (C) Cell cycle profiles and western blot results for assays of MDA-MB-436 cells.