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

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