Figure 1. ERRα upregulates SCT expression in N-42 cells.

(A) The 5′ upstream sequence of the SCT gene contains a ERE-half site. The nucleotide sequences (180 bp) upstream of the start codon of the mouse, human and rat SCT 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 SCT 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 SCT measured by real-time PCR were normalized with mouse GAPDH 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 SCT measured by real-time PCR were normalized with mouse GAPDH 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).