Fig 1: Diminished RA signaling in PEX tissues. (A) Expression of CRBP1, CRABP2, RARA and RXRA mRNA in iris (n = 42) and ciliary body specimens (n = 22) from normal human donors (control) and donors with PEX syndrome (n = 24) using real-time PCR technology. Expression levels were significantly reduced in PEX specimens compared to control specimens. The relative expression levels were normalized relative to GAPDH and are represented as mean values ± SD. (B) Western blot analysis of CRBP1 protein expression in iris and ciliary body tissue from normal donors (control, n = 4) and patients with PEX syndrome (n = 4). Protein expression is normalized to the house-keeping gene ß-actin and is expressed as percent of the expression in controls. (C) Western blot analysis of RBP4 protein in serum and aqueous humor samples from cataract patients (control, n = 4) and patients with PEX syndrome (n = 4). RBP4 protein is normalized to total protein content and is expressed as percent of controls (data represent mean values ± SD of eight samples; *P < 0.05; **P < 0.01 ***P < 0.001). (D) Real-time PCR analysis of RXRA, LOXL1, ELN, FBN1 and TGFB1 mRNA in hTCFs (n = 4) and TM cells (hTMC, n = 4) transfected with RXRA-specific siRNA or scrambled control siRNA; expression levels were normalized relative to GAPDH and expressed relative to mock-transfected controls (dashed line; *P < 0.05; **P < 0.01; ***P < 0.001). (E) Real-time PCR analysis of RXRA, LOXL1, ELN, FBN1 and TGFB1 mRNA in hTCF and hTMC without and with stimulation by 2.5 µm RA for 48 h; expression levels were normalized relative to GAPDH and expressed relative to unstimulated controls (dashed line; *P < 0.05; **P < 0.001; ***P < 0.0001).
Fig 2: Identification and quantification of differentially expressed proteins by iTRAQ. The RALBP-1, RBP-1, and IRBP were identified as differentially expressed visual cycle-associated proteins among the untreated rd12, treated rd12, and normal control C57BL/6J eyes. RALBP-1, RBP-1, and IRBP were remarkably reduced in the untreated rd12 mice (<0.7×) compared to those of C57BL/6J sample. In the treated rd12 eyes, RALBP-1 was increased to 6-fold of the untreated rd12 level, although it was still lower than the normal level (0.66-fold); RBP-1 was increased to 2-fold of the untreated rd12 level and 1.50-fold of the normal level. IRBP level was still lower in the treated rd12 eyes.
Fig 3: Western blot analysis of differentially expressed proteins. (a) The expression of RALBP-1 and RBP-1 was weak in the untreated rd12 eyes and appeared to increase significantly following gene therapy to levels similar to those present in normal wild-type control eyes. (b) and (c) show the relative grayscale of RALBP-1 and RBP-1 compared to the wild-type values.
Fig 4: Expression of specific HSC markers in hypertrophy areas. (a–d) Desmin labelling of 9 weeks SD- (a) and CDAHFD-fed mice (b–d) liver slices. Overlap of DAPI, RF and desmin signals (a,c,d) or DAPI and desmin signal (b). Arrowheads: desmin-positive cells. Stars: desmin- and retinoid-positive cells. (e–h) cRBP1 labelling of 9 weeks SD- (e) and CDAHFD-fed mice (f–h) liver slices. Overlap of DAPI, RF and cRBP1 signals (e,g,h) or DAPI and cRBP1 (f).
Fig 5: Allele-specific transcription factor binding at the genomic region surrounding rs7173049.(A)Transcription factor binding sites overlapping SNP rs7173049 (marked in red) regions (±15 bp) as predicted by the PROMO bioinformatics program. Differential binding to sequences containing the protective G allele (rs717-G) compared to sequences containing the A allele (rs717-A) was only predicted for T3R-beta1 (THRß1). Each gray/orange box indicates one transcription factor: GR-alpha, ER-alpha, XBP-1 and YY1. (B) Supershift assays with 31 bp biotinylated DNA probes containing rs7173049 G allele (rs717-G), nuclear extracts from hTCFs and specific antibodies against candidate transcription factors RXRa-1,2,3, beta (RXRß) and gamma (RXR?), RARa/ß/? and thyroid hormone receptor alpha/beta (THRa/ß) showing disruption of the shifted DNA–protein complex (solid arrow) and formation of supershifted bands (dotted arrows) with the THRa/ßantibody. Dotted black line indicates a cut and junction of two parts of the same membrane. (C) Supershift assays with THRa/ß antibody attenuated the shifted DNA–protein complex (solid arrow) and produced distinct supershifted bands (dotted arrows) with DNA probes containing the protective allele G of rs7173049 (rs717-G). No supershifted bands were detected with probes containing the A allele (rs717-A). Bands which were not competitively inhibited by unlabeled oligonucleotides (arrowhead) were considered unspecific. (D) Increasing amounts of recombinant THRß protein were used in positive control experiments to show a specific interaction with the DNA probe containing the protective G allele of rs7173049 (rs717-G). DNA–protein complexes (arrows) presumably represent thyroid hormone receptor (THR) monomers and homodimers. (E) Real-time PCR analysis of THRB, CRBP1, CRABP2, RARA and RXRA mRNA in hTCFs (n = 4) transfected with THRB-specific siRNA or scrambled control siRNA; expression levels were normalized relative to GAPDH and expressed relative to mock-transfected controls (dashed line; (*P < 0.05; **P < 0.01 ***P < 0.001).
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