MEHP upregulates expression of CRH and COX-2 in primary cultures of human cytotrophoblast.

<p>Primary cultures of term cytotrophoblast were exposed to different concentrations of MEHP for 24 hours with DMSO as the vehicle control. (<b>A</b>) Trypan blue exclusion test of cell viability for primary cytototrophoblast exposed to MEHP at concentrations as indicated (N = 3 independent experiments). (<b>B</b>) Representative gel patterns and analysis in whole cell lysates from three independent term placentas by Western blot assay with use of antibody as indicated. (<b>C</b>) Total RNAs were extracted and RT-qPCR was performed for assessment of CRH and COX-2 mRNA levels with normalization to GAPDH mRNA level. Bars represent the average of relative mRNA levels and error bars represent standard deviation from experiments performed in three independent term placentas. * p < 0.05; ** p < 0.01.</p

NIK knockdown attenuates MEHP-induced upregulation of <i>CRH</i> and <i>COX-2</i> in the human placenta.

<p>Primary term cytotrophoblast were incubated with siRNAs targeting NIK (siNIK) for 24 hrs, and then treated with MEHP for an additional 24 hrs. Scramble siRNA (Scr-siRNA) was used as the non-targeting control. Total RNAs were extracted and RT-qPCR was performed to determine mRNA levels of CRH (<b>A</b>), COX-2 (<b>B</b>), or NIK (<b>C</b>) (N = 3 independent experiments). * p < 0.05 (compared to DMSO). ** p< 0.01 (compared to DMSO). ## p < 0.01 (compared to Scr-siRNA). (<b>D</b>) Representative gel pattern of Western blot analysis on whole cell lysates from three independent experiments. (<b>E</b>) IF staining was performed to directly assess intracellular distribution of RelB. These images represent data obtained from three independent placentas. Original magnification, 200Χ.</p

The effects of MEHP exposure on interaction of RelB/p52 with the <i>CRH/COX-2</i> gene promoters.

<p>Term cytotrophoblast were exposed to MEHP for 24 hrs at concentrations as indicated. ChIP assays were performed to determine occupancy of RelB/p52 at <i>CRH</i> (<b>A</b>) and <i>COX-2</i> (<b>B</b>), and also occupancy of RelA at the <i>CRH</i> (<b>C</b>) and <i>COX-2</i> (<b>D</b>) gene promoter. Fold enrichment was derived with normalization to a human DNA a satellite, a non-coding DNA sequence to which no transcriptional factors should bind. Rabbit IgG was used as a non-specific control. The bars indicate the average of fold enrichment with error bars representing the standard deviation from three independent experiments. * p < 0.01.</p