Effect of CDCA on hypoxic target genes.

<p>(A) Experimental scheme. HepG2 cells were serum starved with medium containing 0.5% FBS for 20 hours prior to CDCA (100 μM or indicated dose) treatment. 6 hours after CDCA treatment, the cells were exposed to 20%, 5% or 0.1% O₂ for the indicated hours. (B) Quantitative RT-PCR analyses of SHP mRNA. The expression level was normalized with the expression level of 18s rRNA. (C) Western analyses for SHP and β-actin. β-actin protein was detected as a loading control. Data shown are representative of three experiments (C) Quantitative RT-PCR analyses of carbonic anhydrase 9 (CA9), phosphoglycerate kinase1 (PGK1), endoplasmic reticulum oxidoreductin 1-like (EROL1), lysyl oxidase (LOX), prolyl 4-hydroxylase, alpha peptide 1 (P4HA1). a, <i>p</i> ≤ 0.1; b, <i>p</i> ≤ 0.05; c, <i>p</i> ≤ 0.01; d, <i>p</i> ≤ 0.001; e, <i>p</i> = 0.197; f, <i>p</i> = 0.724.</p

ERRα upregulates SCT expression in N-42 cells.

<p>(A) The 5′ upstream sequence of the <i>SCT</i> gene contains a ERE-half site. The nucleotide sequences (180 bp) upstream of the start codon of the mouse, human and rat <i>SCT</i> gene are shown. The first nucleotide of the start codon (ATG) is assigned as +1. The consensus sequences of the putative ERE-half site are highlighted in red and underlined. The mutation sites are indicated by the boxes (B) Effects of over-expressing ERRs in N-42 on the mouse <i>SCT</i> promoter. mSCTP (0.5 µg) and various amounts of the 3 isoforms of ERR/pcDNA3 (0, 0.5, 1.0 and 2.0 µg) were cotransfected into N-42 cells. Total DNA was adjusted to 2.5 µg by pKS⁺. *p<0.05; **p<0.001, compared with mSCTP (0.5 µg). (C) Effects of over-expressing ERRα on the mouse SCT mRNA levels in N-42 cells. The mRNA levels of mouse <i>SCT</i> measured by real-time PCR were normalized with mouse <i>GAPDH</i> levels. *p<0.05; **p<0.001, compared with control (ERRα – 0 µg). (D–E) Effects of endogenous silencing of mouse ERRα on (D) mouse SCT promoter and (E) mRNA levels in N-42 cells. The mSCTP (1.0 µg) was co-transfected with various amounts of siERRα-1 and siERRα-2 (1.0 and 2.0 µg), pSilencer or siControl into N-42 cells. The mRNA levels of mouse <i>SCT</i> measured by real-time PCR were normalized with mouse <i>GAPDH</i> levels. Data represent the mean ± SEM of three experiments performed in duplicates. *p<0.05; **p<0.001, compared with mSCTP – 1.0 µg. (B) *p<0.05; **p<0.001, compared with control (pSilencer – 2.0 µg). (F) Western blot analysis of ERRα protein in N-42 cells transfected with (1) pSilencer (2.0 µg), (2) siERRα-S1, (3) siERRα-S2 and (4) siControl (2.0 µg). The GAPDH western blot was used as the loading control. (G) Mutation analysis of ERE-half site. Four mutants (M1–M4, 0.5 µg) were cotransfected with pKS⁺ or ERRα expression vector (2.0 µg). ⧫p<0.05; ERRα cotransfected promoter compared with the same construct that transfected with pKS⁺ (0.5 µg).</p

Effect of saline and ANGII on the binding of ERRα to the mouse SCT promoter.

<p>(A) ChIP assay showing the relative occupancy of the immunoprecipitated ERRα at the mouse <i>SCT</i> promoter in N42 cells after saline and ANGII treatment for 8 h in N-42 cells. Data was calculated using the equation: (Ct^mock–Ct^specific) and normalized with Input, which was defined as 1.00. A mock immunoprecipitation using rabbit anti-IgG and without using antibody were carried out as negative control. Data represent the mean ± SEM of three experiments.*p<0.05; **p<0.001, compared with the control. (B) EMSA using the ERE-half site as the oligo. Left: Nuclear extract of mouse hypothalamus (10 µg), pcDNA (control), and <i>in vitro</i> translated ERRα protein were pre-incubated with the oligo for 15 minutes at room temperature. Arrows indicate specific protein-DNA complexes that contain ERRα (Complex I) and unknown protein (complex II). Middle: Hypothalamic nuclear extract of control or water deprived or saline-drank mice were used (n = 4/group) in EMSA. Right: Supershift assay of ERRα proteins. Antibodies (2 µg) specific for ERRα and AP1 (Santa Cruz) proteins were pre-incubated with the nuclear extract (10 µg) for 20 min at room temperature. Arrows indicate specific protein–DNA complexes that contain ERRα (Complex I), non-specific complex (Complexes II) and ERRα supershift (SS). (C) ChIP assay showing the in vivo binding of ERRα on mouse <i>SCT</i> promoter in mouse hypothalamus after water deprivation and saline treatment (left) and ICV-ANGII injection in mice (1 and 4 h) (right).</p

Effect of CDCA on HIF-1α expression.

<p>After starvation for 20hr, HepG2 cells were pretreated with CDCA (100 μM or indicated dose) for 6 hours then exposed to 20%, 5% or 0.1% O₂ for the indicated hours. (A) Western analyses for HIF-1α, 14-3-3γ and β-actin proteins. 14-3-3γ and β-actin proteins were detected as loading controls. (B) Quantitative RT-PCR of HIF-1α mRNA. (C and D) Western analyses of HIF-1α protein. HepG2 cells which were serum starved with medium containing 0.5% FBS for 20 hours prior to stimulation with MG132 (10 μM) and/or CDCA. 6 hours after treatment, the cells were exposed to 20% or 5% O₂ for 4 hours. Ubiquitinated and original HIF-1α proteins are indicated. HDAC1 protein or β-actin protein were examined in order to verify equal loading. (D) 20 μg of MG132 untreated total cell extracts and 5 μg of MG132 treated total cell extracts are loaded, respectively.</p

Effects of SHP or GW4064 on HIF-1α expression.

<p>(A and B) HepG2 cells were transfected with an empty vector or pcDNA3/HA-SHP. 18 hours after transfection, the cells were serum starved with medium containing 0.5% FBS for 20 hours. The cells were treated DMSO or 100 μM of CDCA for 6 hours in the absence or presence of MG132 and then exposed to 20%, 5% or 0.1% O₂ for 4 hours. 30 μg of total cell extracts are loaded for western analyses. (C to E) After starvation for 20hr, HepG2 cells were pretreated with GW4064 (GW) (5 μM or indicated dose) for 6 hours then exposed to 20%, 5% or 0.1% O₂ for the indicated hours. (C and E) qRT-PCR analyses of SHP or HIF-1α respectively. The expression level was normalized with the expression level of 18s rRNA. a, <i>p</i> ≤ 0.1; b, <i>p</i> ≤ 0.05; c, <i>p</i> ≤ 0.01; d, <i>p</i> ≤ 0.001. (D) Western analyses of HIF-1α, SHP and β-actin. (F) Western analyses of HIF-1α and β-actin in HepG2 cells which were treated with indicated doses of GW4064 and MG132 as described above. Ubiquitinated and original HIF-1α proteins are indicated. β-actin protein were examined in order to verify equal loading.</p

TGF-β1 signaling regulates steroidogenic gene expression, affecting testicular testosterone levels in mice.

<p>(<b>A</b>) Decreased Tgfbr2^fl°^x allele in purified primary Leydig cells isolated from mice harboring the Cyp17iCre transgene. The genomic DNA isolated from primary Leydig cells of Tgfbr2^flox/flox and Tgfbr2^flox/flox Cyp17iCre mice was amplified for Tgfbr2 intron region containing the LoxP site. A pair of β-actin primers was used as the control for the amount of genomic DNA. (<b>B</b>) Decreased TGF-β1-mediated repression of steroidogenic gene expression with Tgfbr2 silencing. Purified primary Leydig cells from the testes of 12-week-old Tgfbr2^flox/flox (n = 6) and Tgfbr2^flox/flox Cyp17iCre (n = 6) mice were treated with 300 µM of 8-Br-cAMP and 2 ng/ml of TGF-β1 for 24 hours, and mRNA expression levels were measured using qRT-PCR. β-actin expression was used as a loading control. The data are presented as the mean ± SEM. **, P<0.01; ***, P<0.01. (<b>C</b>) Testicular testosterone levels were measured by RIA in the testes of 5 week-old Tgfbr2^flox/flox and Tgfbr2^flox/flox Cyp17iCre mice. (<b>D</b>) Total protein (100 µg) from the testes of 5 week-old Tgfbr2^flox/flox and Tgfbr2^flox/flox Cyp17iCre mice was subjected to western blot analysis for protein levels of steroidogenic genes. The relative level of each protein/GAPDH was quantified by densitometric analysis using Image J software. In panels C and D, the data are presented as the mean ± SD (n = 10). **, P<0.01.</p

Effect of CDCA on de novo synthesis of HIF-1α protein.

<p>(A) Experimental scheme. After serum starvation for 20 hr, HepG2 cells were pretreated with DMSO or CDCA (100μM), 30 min prior to MG132 (10 μM) treatment. (B) Western analyses of HIF-1α and β-actin proteins (three western blots are shown). (C) Experimental scheme. HepG2 cells were pretreated with CHX (10 μg/ml, 3 hr), then the culture media were replaced with fresh media containing MG132 (10 μM) and/or CDCA (100 μM) as indicated. (D) Western analyses of HIF-1α and β-actin proteins (three western blots are shown). Quantification of western analyses. The intensities of HIF-1α and β-actin bands marked with bars were measured using Image J software. The y-axis indicates the relative band intensities of HIF-1α protein to 0 hr. The band intensities of HIF-1α protein were normalized by β-actin protein. The x-axis indicates the hours for MG132 treatments. <i>p</i> values between band intensities of CDCA-treated and untreated samples are shown. a, <i>p</i> ≤ 0.1; b, <i>p</i> ≤ 0.05; c, <i>p</i> ≤ 0.01; d, <i>p</i> ≤ 0.001; e, <i>p</i> = 0.251.</p

Effects of water deprivation, saline dinking and central ANGII administration on mouse <i>ERRα</i> expressions in mouse brain.

<p>Mice were water deprived, provided with hypertonic saline (2%) for 1 or 5 d, or centrally injected with ANGII peptide. The mRNA levels of mouse <i>ERR</i>α in (A) the whole mouse brain or (C) the isolated osmosentitive brain regions SFO, OVLT, MnPO and PVN, were measured by real-time PCR were normalized with mouse <i>GAPDH</i> levels. Data are expressed as the mean ± SEM (n = 10/group). *p<0.05; **p<0.001, compared with control. (B) Immunohistochemical staining showing ERRα immunoreactivities in the SFO, MnPO, OVLT and PVN of the WT mouse brain. Negative control was done by using 1× PBS instead of the primary anti-ERRα antibody. Bars, 6 µm.</p

Effects of saline and ANGII treatments on <i>SCT</i> promoter activity and gene expression in N-42 cells.

<p>(A) Osmolality of culture medium after treatment of different concentrations of saline (0, 50, 100, 200, 300, 400 mM) for 8 h in N-42 cells. (B) Percentage of cells surviving after saline treatments with medium of various concentrations (0, 50, 100, 150, 200, 300, 400 mM) for 8 h in N-42 cells. (C) Cells were transfected with mSCTP (2.0 µg) for 2 d and treated with various saline concentrations (25, 50, 100 mM) for different times (2, 4, 8 h) or ANGII (10⁻¹⁰ to 10⁻⁷ M) for 8 h. Data represent the mean ± SEM of three experiments performed in triplicates. (D and E) Cells were treated with various saline concentrations (25, 50, 100 mM) for different times (2, 4, 8 h) and RNA was extracted afterwards. The mRNA levels of mouse <i>SCT</i> (D) and <i>S14</i> (E) were normalized with mouse <i>GAPDH</i> levels. Data represent the mean ± SEM of three experiments performed in duplicates.*p<0.05; **p<0.001, compared with the respective control.</p

TGF-β1/ALK5 signaling represses cAMP-induced steroidogenic gene expression in Leydig cells.

<p>(<b>A and B</b>) The culture medium of purified mouse primary Leydig cells treated with 300 µM of 8-Br-cAMP and 5 ng/ml of TGF-β1 (A) and R2C cells treated with vehicle or 5 ng/ml of TGF-β1 (B) for 24 hours was collected for the measurement of testosterone levels by RIA. (<b>C and D</b>) The expression levels of steroidogenic genes in primary Leydig cells (C), which were treated with 300 µM of 8-Br-cAMP, 2.5 ng/ml of TGF-β1 and 10 µM SB431542 for 24 hours, and R2C cells (D), which were treated with 5 ng/ml of TGF-β1 for 24 hours, were analyzed by qRT-PCR. (<b>E</b>) The expression level of Tgfbr2 and Tgfbr1 was analyzed using total RNAs from primary Leydig, R2C and MA-10 cells by RT-PCR. (<b>F</b>) MA-10 cells were transiently transfected with the ALK5 (TD; constitutively active form) expression plasmid, along with an indicated reporter of the natural promoter, in medium containing 5% charcoal stripped FBS. Twenty four hours after transfection, the cells were treated with 300 µM of 8-Br-cAMP for 24 hours and harvested for luciferase assay. The pSV-β-gal expression plasmid was used as a control for transfection efficiency. The data are presented as the mean ± SEM of at least three independent experiments. **, P<0.01; ***, P<0.001; ns, not significant.</p