Table 1.
Oligonucleotide primer sequences for quantitative real-time PCR.
Figure 1.
AR and Kitl mRNA levels decreased in Nur77−/− mouse ovaries.
RNA was collected from 20-week-old Nur77+/+ and Nur77−/− female mouse ovaries and reverse transcribed for real-time PCR. AR (A), Kitl (B) and Nur77 (C) mRNA levels of Nur77−/− mouse ovaries (n = 5) versus Nur77+/+ mouse ovaries (n = 5) were measured and expressed as relative quantities (*P<0.05, **P<0.01).
Figure 2.
Effects of Nur77 overexpression and knockout on mouse AR and Kitl expression. A:
Nur77+/+ mGCs were infected with Ad-LacZ or Ad-Flag-Nur77 at the indicated MOI for 48 h. AR and Kitl mRNA levels were measured by real-time PCR and shown as a ratio over control (Ad-LacZ). B: AR protein levels were measured by western blot analysis. C: Real-time PCR analysis of AR and Kitl mRNA expression in Nur77+/+ and Nur77−/− mGCs from 3-week-old mice. D: Western blot analysis of AR protein expression in Nur77+/+ and Nur77−/− mGCs from 3-week-old mice. The results are an average of three independent experiments performed in triplicate (*P<0.05, **P<0.01).
Figure 3.
Nur77 binds to the mouse AR promoter and stimulates its activity.
A: The results of ChIP-PCR amplification are shown using primers against the mouse AR promoter region. PCR products were separated by agarose gel electrophoresis. B: HEK293T cells were transfected with pCMV-Nur77 or pCMV-empty vector plasmid and cotransfected with the mAR promoter-luciferase construct using the Nanofectin transfection reagent. After 48 h, luciferase assays were performed and normalized by constitutive Renilla luciferase. C: mGCs from Nur77+/+, Nur77+/− and Nur77−/− mice were transfected with mAR promoter-luciferase construct using Lipofectamine 2000 reagent. After 48 h, luciferase assays were performed and normalized by constitutive Renilla luciferase (n = 3; *P<0.05, **P<0.01).
Figure 4.
Effects of Nur77 overexpression and silencing on human AR and KITLG expression in KGN cells. A:
KGN cells were infected with Ad-LacZ or Ad-Flag-Nur77 at the indicated MOI for 48 h, and AR and KITLG mRNA levels were measured by real-time PCR and shown as a ratio over control (Ad-LacZ). (n = 3; *P<0.05, **P<0.01). B: Western blot analysis of AR protein expression in KGN cells treated with Ad-LacZ or Ad-Flag-Nur77 at the indicted MOI for 48 h. A: Real-time PCR analysis of AR and KITLG mRNA expression levels in KGN cells infected with Ad-LacZ or Ad-siNur77 (MOI = 50 and 100) for 48 h (n = 3; *P<0.05, **P<0.01). B: Western blot analysis of AR protein expression in KGN cells treated with Ad-LacZ or Ad-siNur77 at the indicated MOI for 48h.
Figure 5.
Nur77 binds to the human AR promoter and enhances its activity.
A: The results of ChIP-PCR amplification are shown using primers against the human AR promoter region. PCR products were separated by agarose gel electrophoresis. B: Quantitative ChIP was performed with an antibody to Nur77 or irrelevant control antibody. Co-precipitating chromatin fragments were analyzed by real time PCR using primer sets that span −2553/−2401 bp, −2584/−2413 bp, −4713/−4526 bp and −4119/−3967 bp in human AR promoter region. Results were normalized to total input (not precipitated) chromatin(n = 3; *P<0.05, **P<0.01). C: ABCD assays were performed using biotinylated or unbiotinylated (competitor) double-stranded AR wild-type (WT) and AR mutant (Mut) oligonucleotides in addition to KGN cell extracts after infection with Ad-LacZ or Ad-Flag-Nur77. D: HEK293T cells were transfected with pCMV-Nur77 or pCMV-empty vector and cotransfected with the hAR promoter-luciferase construct using the Nanofectin transfection reagent. Forty-eight hours after transfection, luciferase assays were performed and normalized by constitutive Renilla luciferase (n = 3; *P<0.05, **P<0.01).
Figure 6.
Nur77 affected the expression of the androgen signaling target gene, Kitl, by regulating AR.
KGN cells were infected with Ad-LacZ or Ad-Flag-Nur77 at 10 MOI, and Nur77+/+ or Nur77−/− mGCs were treated with androstenedione (A, C) or the androgen signaling antagonist, flutamide (B, D). Cellular RNA was collected, and real-time PCR analysis showed Kitl expression levels with different treatments. The results are shown as a ratio over control (n = 3). Values with different superscripts (a, b, c, d) are significantly different (p<0.05) from each other.