Figures & data
Figure 1. Dose-response effect of PCBs on AR transcriptional activity in HEK293 cells transiently cotransfected with pCMV-hAR, GRE2E1bLuc, and pgL4hR and treated with DHT 10−7 M, or with the PCB mixture at 10−6, 10−7 and 10−8 M, or with ethanol (EtOH) as negative control. EtOH, ethanol; PCB, polychlorinated biphenils; AR, androgen receptor.
Figure 2. Enhancement of transcriptional activity of AR by Jarid1b in HEK293, GN11, and NSC34 cells. Cells cotransfected with pCMV-hAR, pcDNA 3.1-hJarid1b-myc-his (or the empty vector, as control), GRE2E1bLuc, and pgL4hRLuc were treated with DHT 10−7 M; the PCB mixture 10−7 M; or ethanol (EtOH). EtOH, ethanol; PCB, polychlorinated biphenils; DHT, dihydrotestosterone; AR, androgen receptor.
Figure 3. Western blot analysis of H3K4me3 levels in HEK293 cells transfected with the plasmid pcDNA 3.1-hJarid1b-myc-his (or the empty vector, as control) overexpressing histone demethylating enzyme Jarid1b. The relative amount of H3K4me3 was normalized by the signal obtained for total H4 with an anti-H4 antibody. EtOH, ethanol; PCB, polychlorinated biphenils; DHT, Dihydrotestosterone; J, Jarid1b
Figure 4. Immunofluorescence analysis of AR nuclear translocation in HEK293 cells transiently cotransfected with the plasmid 3.1-hJarid1b-myc-his following DHT or PCB treatment. Primary antibodies against AR and pcDNA 3.1-hJarid1b-myc-his were used to detect them. Secondary conjugated antibodies were used to detect AR (green channel) or Jarid1b (red channel). Images were obtained at either 20× or 32× magnification.
Figure 5. Transcriptional activity of different Poly Q variant of AR in HEK 293 cells cotransfected with plasmids (pCMV3.AR/Q12 or pCMV3.AR/Q23 or pCMV3.AR/46) coding for a AR with different CAG repeat length (12 PolyQ, 23 PolyQ or 46 PolyQ) and pcDNA 3.1-hJarid1b-myc-his (or an empty vector). Cells were treated with ETOH, DHT 10–7 M or the PCB mixture 10–7 M. EtOH, ethanol; PCB, polychlorinated biphenils; DHT, dihydrotestosterone.
Figure 6. In silico analysis on the binding sites for hormone receptors and for Jarid1b present on AR promoter. The computer platform MatInspector (available on www.genomatix.de/online) was used. A stretch of the AR gene sequence spanning from about 5000 bp before the transcription start site (TSS) to about 2800 bp after TSS was examined. ARE, Androgen Responsive Element; GRE, Glucocorticoid Responsive Element; ERE, Estrogen Responsive Element; AhRE Element responsive to AhR (the aryl hydrocarbon receptor) and Jarid1b (also known as PLU1) binding sites. Start and End rows represent the relative position of the potential binding site relative to the sequence analyzed. At the right side of the figure the plasmids used in figure above are aligned for the sequences. The position of binding site for Jarid 1b and AhRE are exactly localized. Bp, base pair; TSS, transcription start sSites; AhRE, element responsive to AhR; ARE, androgen responsive element; GRE, glucocorticoid responsive element; ERE, estrogen responsive element; AR, androgen receptor.
Figure 7. Transcriptional activity of AR by different length AR promoters on HEK 293. The cells were cotransfected with pcDNA 3.1-hJarid1bmyc-his (or the same empty vector), pCMV human androgen receptor, pgL7.0, pgL3.5, and pgL1.8 (coding for AR promoters of differential length). Cells were treated with ETOH, DHT 10–7 M or the PCB mixture 10–7 M. EtOH, ethanol; PCB, polychlorinated biphenils; DHT, dihydrotestosterone; bp, base pair; AhRE, element responsive to AhR; AR, androgen receptor; J: Jarid.
