Common Variation in EDN1 Regulatory Regions Highlights the Role of PPARγ as a Key Regulator of Endothelin in vitro

Figures

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  Figure 1. Common variants in EDN1 regulatory regions show different activities depending on the genotype. (A) Schematic view of the EDN1 gene with the location of the SNPs and the predicted transcription factors to interact with them. (B) Comparison of the different genotypes of rs397751713 show increased activity in the A/A genotype and A/- when compared to the commonest genotype –/– (n = 6). (C) Comparison of the different genotypes of rs2859338 show increased G/G genotype activity and reduced activity in A/A compared with the commonest genotype A/G (n = 4). Data are represented as box plots depicting the quartiles and dots representing technical replicates. A Kruskal-Wallis test with a Dunn's correction was used for statistical analysis (****p > 0.0001). EDN1, Endothelin-1.

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  Figure 2. The KD of PPARγ, KLF4, and VDR increase the mRNA levels of EDN1. Data are shown as box plots depicting the quartiles (n = 3). Dots represent technical replicates. A Kruskal-Wallis test with a Dunn's correction was used for statistical analysis (*p > 0.05, ****p > 0.0001). EDN1, Endothelin-1; PPARγ, peroxisome proliferator-activated receptor γ; KLF4, Krüppel-Like Factor 4; VDR, vitamin D receptor.

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  Figure 3. Knock-Down efficiency for KLF4 (A), PPARg (B), and VDR (C). Data are shown as box plots representing median ± quartiles (n = 4). Dots represent technical replicates. A Student's t-test was used for statistical analysis (****p > 0.0001). PPARg, peroxisome proliferator-activated receptor g; KLF4, Krüppel-Like Factor 4; VDR, vitamin D receptor.

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  Figure 4. KD experiments demonstrate that PPARγ binds in rs397751713. (A) Comparison of the luciferase activity in the homozygous genotypes of rs397751713 after different treatments, siKLF4 and Mock show the same pattern while siPPARγ increases the activity of both genotypes to similar levels (n = 3). (B) Comparison of the luciferase activity in the homozygous genotypes of rs2859338 after Mock or siVDR treatment shows the same pattern. Data are shown as box plots representing median ± quartiles. Dots represent biological replicates. PPARγ, peroxisome proliferator-activated receptor γ.

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  Figure 5. ChIP-qPCR using an anti-PPARγ antibody demonstrates that low amounts of PPARγ binds around rs397751713. (A) Full plot with the input chromatin used in the assay, a positive control (H3), and a Rabbit IgG. (B) Comparison between the control IgG and PPARγ amplification. Data are shown as box plots representing median ± quartiles. Dots represent technical replicates from two biological replicates. A Student's t-test was used for statistical analysis (**p > 0.01). PPARγ, peroxisome proliferator-activated receptor γ.

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  Figure 6. Silencing PPARγ increases ET-1 production in HeLa and PAECs while VDR does only in PAECs and KLF4 in HeLa. (A) Representative immunofluorescences for HeLa cells. (B) Quantification of ET-1 production using immunofluorescence in HeLa, treatment with siKLF4 and siPPARγ increases ET-1 fluorescence (n = 4). (C) Representative immunofluorescences for PAECs. (D) Quantification of ET-1 production using immunofluorescence in PAECs, treatment with siPPARγ and siVDR show the highest increase in ET-1 fluorescence, while siKLF4 has a slight increase (n = 6). Data are represented as box plots depicting the quartiles and dots representing single cells measured. A Kruskal-Wallis test with a Dunn's correction was used for statistical analysis (*p > 0.05, ****p > 0.0001). PPARγ, peroxisome proliferator-activated receptor γ.

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  Figure 7. Quantification of ET-1 by sandwich ELISA. (A) Raw values. (B) After normalizing against the total protein from the well. Data are shown as box plots representing median ± quartiles. Dots represent biological replicates (n = 4). A Kruskal-Wallis test with a Dunn's correction was used for statistical analysis.