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. 2017 May 23;8:15466. doi: 10.1038/ncomms15466

Figure 9. RXRα influences LOXL1 expression by functioning as a transcriptional repressor.

Figure 9

(a) ChIP assay for RXRα binding at rs11638944 (SNP12)-containing region of LOXL1 in heterozygous hTCF cell lines (n=2) using two different antibodies against RXRα (1: D-20, 2: F-1), histone H3 (positive control) and non-immune IgG (negative control); input represents total chromatin applied for immunoprecipitation. DNA isolated from immunoprecipitated complexes was analysed on 2% agarose gel (left) and by qPCR (right) with primers specific for the SNP12 region producing a 148 bp PCR fragment (arrow). Data are expressed as per cent of input (Lane 1: hTCF 1, lane 2: hTCF 2, lane M: DNA marker, lane N: primer control without chromatin). (b) Dual luciferase reporter assays using reporter plasmids containing risk (12R) and non-risk (12N) genotypes of SNP 12 alone or co-transfected with RXRα-specific siRNA in human Tenon's capsule fibroblasts (hTCF) compared with the basal LOXL1 promoter activity. Results are expressed as the ratio of Firefly luciferase to Renilla luciferase; the transcriptional activity of the empty pGL4.10 vector was set at 100% (data represent mean values±s.d. of three independent experiments; *P<0.005; unpaired two-tailed Student's t-test). (c) Western blot analysis of RXRα expression in cytoplasmic and nuclear protein fractions of human Tenon's capsule fibroblasts (hTCF), trabecular meshwork cells (hTMC), nonpigmented ciliary epithelial cells (hNPEC) and aortic smooth muscle cells (hASMC) at ∼60 kDa (C, cytoplasmic fraction; N nuclear fraction). (d) Real-time PCR analysis of RXRα and LOXL1 mRNA in hTCF cell lines (n=4) transfected with RXRα-specific siRNA or scrambled control siRNA; expression levels were normalized relative to GAPDH. (e) Real-time PCR analysis of RXRα and LOXL1 mRNA without and with stimulation by 2 μM all trans-retinoic acid for 48 h in hTCF and hASMC; expression levels were normalized relative to GAPDH (data represent mean values±s.d.; *P<0.05, **P<0.001, ***P<0.0001; unpaired two-tailed Student's t-test.