Figure 5.

Knockdown of necdin in GT1-7 cells decreases GnRH mRNA and gene expression. (A) GT1-7 cells were either mock-transfected or transfected with non-targeting (control), cyclophilin B (CyB) or necdin (Ndn) siRNA duplexes. Specific knockdown of necdin and cyclophilin B proteins was detected by immunoblot. (B) GT1-7 cells were transfected as in (A) followed by RNA isolation. GnRH was measured by quantitative RT–PCR and data are shown as pg GnRH mRNA when normalized to CyB mRNA. Results are the means of three experiments performed in triplicate and *indicates significantly different from control by Student’s t-test (P < 0.05). (C) GT1-7 cells were co-transfected with control or Ndn siRNA duplexes and either GnRH enhancer reporter or mutant GnRH enhancer reporter. Experiments were performed in quadruplicate and repeated at least three times. Results shown are mean ± SEM. One-way ANOVA and Tukey–Kramer HSD compared the means. *P < 0.05.