Article Figures & Data
Figures
- Fig. 1.
Strain-dependent expression and inducibility of ALDH1A isoforms. Normalized expression of ALDH1A7 (A) and ALDH1A1 (B) mRNA in control, PB-, or PCN-treated RR and rr rat livers. Levels are expressed as scatter plots and means ± S.D. from four animals, each sample with two technical replicates. Cytosolic and total protein levels of ALDH1 protein (C) and activity (D) in control, PB-, or PCN-treated RR and rr rat livers. Statistically significant differences (P < 0.05) compared with the respective control group or between the strains are marked by * and # signs, respectively. The data in (A, B, and D) are also shown relative to the control RR values, set at 1.0, and expressed as means ± S.D. of -fold change below the x-axis.
- Fig. 2.
Similar expression of CYP2Bs and NRs in both rat substrains. Normalized expression of CYP2B1 (A) and CYP3A23 (B) mRNA in control, PB-, or PCN-treated RR and rr rat livers. (C) Comparison of hepatic CAR and PXR mRNA expression between control rat strains. The ratio of target gene to β-actin mRNA expression (y-axis) are shown as scatter plots and means ± S.D. from four animals, each sample with two technical replicates. These normalized levels are also depicted as relative to the mean ± S.D. of the control RR strain, set at 1.0, and indicated below the x-axis. Statistically significant differences (P < 0.05) compared with the respective control group or between the strains are marked by * and # signs, respectively. (D) Expression of CAR and PXR proteins in control, PB- or PCN-treated RR and rr rat livers.
- Fig. 3.
Strain-dependent activation of ALDH1A7 promoter constructs by NRs. Luciferase activity of the full-length RR-ALDH1A7 (gray columns) and rr-ALDH1A7 (black columns) constructs cotransfected with empty (−) or indicated CAR or PXR expression vectors (+), and treated with DMSO (ligand −) or selective CAR or PXR activators (ligand +). Positive control plasmids were PBREM-tk-luc for CAR and XREM-3A4-luc for PXR (white columns). Data are means ± S.D. from three independent transfections and expressed relative to the empty vector (set at 1.0). *Denotes a statistically significant difference from the empty vector at P < 0.05.
- Fig. 4.
Basal and NR-elicited activation of proximal ALDH1A7 promoter constructs. (A) Basal luciferase reporter activity from RR-ALDH1A7 and rr-ALDH1A7 longer (−1571/+5 bp) and shorter (−461/+5 bp) promoter fragments. #Denotes a statistically significant different from the pGL3-Basic construct at P < 0.05; (B) NR-dependent luciferase activity from RR-ALDH1A7 and rr-ALDH1A7 longer and shorter promoter fragments in cotransfection assays with empty or indicated NR expression vectors. ns, no statistically significant differences between constructs with empty or NR expression vectors.
- Fig. 5.
Polymerase II binding to RR-ALDH1A7 and rr-ALDH1A7 proximal promoters. Both strains were dosed with saline (C) or PB for 3 hours; liver nuclear fractions were isolated and crosslinked, and RNA polymerase-associated DNA was analyzed by ChIP assay as detailed in Materials and Methods. The amount of recovered DNA is shown relative to the negative control (nc) region (set at 1.0). Data are scatter plots from two independent experiments, each sample with two technical replicates.
- Fig. 6.
RXRα binding to putative CAR binding sites in the distal regions of RR-ALDH1A7 (A) and rr-ALDH1A7 (B) promoters. Both strains were dosed with saline (C) or PB for 3 hours, and liver nuclear fractions were isolated and crosslinked, and RXRα- associated DNA was analyzed by ChIP assay as detailed in Materials and Methods. The amount of recovered DNA is shown relative to the negative control region (set at 1.0). Data are scatter plots from two independent experiments, each sample with two technical replicates.
Additional Files
Data Supplement
- Supplemental Data -
Supplementary Table 1 - Primers for ALDH1A7 cloning.
Supplementary Table 2 - Primers for ChIP assay.
Supplementary Table 3 - Predicted transcription factor sites in the RR and rr ALDH1A7 upstream sequences.
Supplementary Table 4 - Predicted nuclear receptor binding elements in the RR and rr ALDH1A7 upstream sequences.
Supplementary Table 5 - Group mean Ct values of -actin, ALDH and CYP mRNAs in the multiple dose study.
Supplementary Figure 1 - Alignment of the ALDH1A7 promoters from the RR and rr strains.
Supplementary Figure 2 - Cytosolic and total ALDH1 protein levels in control, PB- and PCN-treated RR and rr rat livers.
Supplementary Figure 3 - Total CAR and PXR protein levels in control, PB- and PCN-treated RR and rr rat livers.
Supplementary Figure 4 - Time course of PB induction in RR and rr rats.
Supplementary Figure 5 - Time course of PCN induction in RR and rr rats.
- Supplemental Data -
In this issue
Strain-Dependent ALDH1A7 Expression in the Rat
