Figure 2

Peroxiredoxin-3 (PRDX3) was downregulated in trophoblastic cells by hydrophobic bile acid instead of promoter hypermethylation.

(A) DNA methylation analysis for detecting PRDX3 promoter methylation level in placentas from normal pregnancies and intrahepatic cholestasis of pregnancy patients. The percentages of methylated DNA were very low in both groups. JAR (B) and HTR8 (C) cells were treated with 5 μM 5-aza-2′deoxycytidine (AZA), a specific inhibitor of DNA methylation for 72 hours. After harvest, the messenger RNA (mRNA) level of PRDX3 was determined by quantitative reverse transcription - polymerase chain reaction (qRT-PCR) assay. (D,E) JAR and HTR8 cells were treated with vehicle (Con), or with μM cholic acid (CA), deoxycholic acid (DCA) and chenodeoxycholic acid (CDCA) for 24 hours. The mRNA level of PRDX3 in JAR (D) and HTR8 (E) was determined by qRT-PCR assay with normalisation to 18s rRNA level. (F,G) JAR and HTR8 cells treated with 0, 50, 100 or 200 μM DCA for 24 hours were subjected to qRT-PCR assays for detecting PRDX3 mRNA expression. (H) JAR and HTR8 cells were treated with 0, 50, 100 or 200 μM DCA for 24 hours, respectively. After harvest, the protein level of PRDX3 in JAR and HTR8 cell was assayed by Western blot. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as an endogenous reference. Full-length blots were presented in Supplementary Fig. S8. (I,J) Villous explants treated with 0, 10, 50 or 100 μM CA/DCA/CDCA for 24 hours were subjected to qRT-PCR assay for detecting PRDX3 mRNA expression (I) and Western blot assay for detecting PRDX3 protein level (J). Data were shown as mean ± standard error of mean. Statistical significance was determined by Mann-Whitney test, Student’s t test or one-way analysis of variance followed by Dunnett’s post-hoc test. (ns: no significance; *p < 0.05, **p < 0.01, ***p < 0.001, versus the control group).