Enhanced at puberty 1 (EAP1) is a new transcriptional regulator of the female neuroendocrine reproductive axis
J. Clin. Invest. Sabine Heger, et al. 117:2145 doi:10.1172/JCI31752 [Go to this article.]

Figure 4
Lentivirus-mediated delivery of EAP1 siRNAs decreases EAP1 mRNA expression both in vitro and in vivo. (A) Diagram of the lentivirus construct (27) used, showing the site of insertion of a U6 promoter–driven shRNA-expressing cassette. (B) Generation of EAP1 sh1 from the U6 promoter–directed transcriptional cassette. (C) Inhibition of EAP1 gene expression in HiB5 hippocampal neural progenitor cells by EAP1 sh1 and -3, measured 48 hours after infection. EAP1 mRNA content was detected by semiquantitative PCR. Bars are mean ± SEM; n = 6–7 wells/group, except in the case of noninfected control (n = 3). *P < 0.05; ***P < 0.01 versus control or LV-infected group. (D and E) Lack of changes in 2ι,5ι oligoadenylate synthetase–1 (OAS1) mRNA abundance after in vitro infection of hypothalamic slices with lentiviral particles carrying EAP1 sh1. The slices were prepared and infected as described in Supplemental Note 7. OAS1 mRNA was measured by semiquantitative PCR 72 hours after infection. (D) PCR products size-fractionated in a 2% agarose gel stained with ethidium bromide. –RT, no reverse transcription product. MM, molecular markers. (E) Densitometric analysis of the gel shown in D. Cyclophilin (Cyclo) mRNA, a housekeeping gene, was used as the normalizing unit. Accordingly, OAS1 mRNA levels are expressed as mean ± SEM (AU) of the OAS1/cyclophilin mRNA ratio calculated for each group (n = 3).