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SUPPLEMENTARY FIGURE LEGENDS
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Supplementary Figure 1. The rs6533526:C>T, c.*454C>T PITX2 variant decreases
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luciferase activity. A) The c.*454C>T variant alters the predicted target sequence for
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hsa-miR-548p. The arrow indicates the position of the mutant nucleotide. The target
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miRNA was predicted using the MicroSNiPer software. The vertical lines and dots in
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the predicted miRNA target sequence indicate perfect Watson-Crick and G:U wobble
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pairings, respectively. Note that the mutant nucleotide forms a perfect Watson-Crik T-A
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pairing with the has-miR-548p seed sequence. B) Scheme of the cDNA constructs used
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to assay c.*454C>T associated activity. The 3’-end of the firefly luciferase coding
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region (1652 nucleotides), cloned into the pMirTarget vector, was coupled with either
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the wild type or mutant PITX2 3’-UTR (nucleotides c.*1 to c.*737). The numbers below
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the cartoon indicate nucleotide positions. C) The influence of the c.*454C>T variant on
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luciferase activity. HEK-293T cells were transfected with either the wild-type or
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c.*454C>T constructs fused to firefly luciferase. After a 48-hour incubation, luciferase
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activity was measured as described in the Materials and Methods section. RFP, encoded
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by the pMirTarget vector, and endogenous LDH were detected by Western blot as
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transfection and loading controls, respectively. The error bars correspond to the SD of
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three independent experiments carried out in triplicate. The asterisks indicate
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statistically significant differences with respect to the control. p values were obtained
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using a one-way ANOVA followed by Tukey’s multiple-comparison test: p<0.05 (*);
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p<0.01 (**); p<0.001 (***).
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Supplementary Figure 2. The rs79691946:C>T, p.(P297S) and p.(G380Rfs*144)
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variants do not alter FOXC1 DNA-binding. (A) The effect of FOXC1 variants on
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DNA-binding specificity was assessed by EMSA. The oligonucleotides corresponding
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to the FOXC1-binding sequence labeled with biotin at the 5'-end were incubated with
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10 µg of nuclear extracts from HEK-293T cells transfected with the indicated variants
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of recombinant FOXC1. The extracts were separated on a 10% nondenaturing
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polyacrylamide gel electrophoresis and were transferred to a positively charged nylon
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membrane (Hybond-N+, Amersham). FOXC1-oligonucleotide complexes were
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visualized by incubation with streptavidin-HRP conjugate and chemiluminescent
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detection (Chemiluminescent EMSA kit, Thermo Scientific). Unlabeled competitor
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oligonucleotides were pre-incubated at increasing concentrations from 0.2 to 10 pmol (1
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to 50-fold excess) with the labeled probe. The arrowhead indicates the position of full-
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length FOXC1-DNA complexes. (B) The percentage of band volume corresponding to
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protein-oligonucleotide complexes was estimated by densitometry analysis as indicated
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in the Materials and Methods section. The error bars correspond to the SD of three
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independent experiments carried out in triplicate. NT: non-transfected cells (negative
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control). The asterisks indicate statistically significant differences with respect to the
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control. p values were obtained using a one-way ANOVA followed by Tukey’s
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multiple-comparison test: p<0.001 (***).
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Supplementary Figure 3. Subcellular localization of FOXC1 variants associated
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with congenital glaucoma. The cDNA constructs encoding the indicated recombinant
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versions of FOXC1 were transiently expressed in HEK-293T cells. The recombinant
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proteins were detected by fluorescent immunocytochemistry with an anti-myc antibody
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(Santa Cruz Biotechnology). Nuclei were visualized using fluorescent DAPI staining.
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Original magnification: X600.
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SUPPLEMENTARY REFERENCES
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prediction of SNP effects on putative microRNA targets. HumMutat 2010; 31: 1223-
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1232.
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5. Thompson JD, Higgins DG, Gibson TJ: CLUSTAL W: improving the sensitivity of
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progressive multiple sequence alignment through sequence weighting, position-
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specific gap penalties and weight matrix choice. Nucleic Acids Res 1994; 22: 4673-
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6. Linding R, Jensen LJ, Diella F, Bork P, Gibson TJ, Russell RB: Protein disorder
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prediction: implications for structural proteomics. Structure 2003; 11: 1453-1459.
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7. Linding R, Russell RB, Neduva V, Gibson TJ: GlobPlot: Exploring protein
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sequences for globularity and disorder. Nucleic Acids Res 2003; 31: 3701-3708.
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8. Chang TH, Huang HY, Hsu JB, Weng SL, Horng JT, Huang HD: An enhanced
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computational platform for investigating the roles of regulatory RNA and for
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identifying functional RNA motifs. BMC Bioinformatics 2013; 14 Suppl 2: S4.
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