Fig. 1
Oxygen glucose deprivation (OGD)/reperfusion promotes delta-secretase expression, and C/EBPs control delta-secretase expression in primary neurons. a OGD increases delta-secretase expression in neurons. Primary cultures of neurons from rat E17 embryos were seeded in a six-well plate. At DIV 7, neurons were treated with OGD for 2 h. After 24 h of recovery, neurons were collected for western blotting and real-time PCR analysis. At least three wells of neurons were tested in each experiment. *P < 0.05. b OGD increases delta-secretase expression in HEK293 cells. After 10 h of OGD treatment, HEK293 cells were recovered for 12 h; mRNAs were extracted and real-time PCR analysis. All real-time PCR experiments were repeated at least three times. β-actin was employed as a loading control. HEK293 cells were seeded and transfected with the various siRNAs. Forty-eight hours post transfection, all cells were given the OGD/recovery treatment. Cells lysates were analyzed by western blotting (middle panels). Knockdown of HSF, C/EBPα, and C/EBPβ was confirmed by western blotting (lower panels). c, d Overexpression of HSF1, C/EBPα, and C/EBPβ by lentivirus transduction of primary neurons increases delta-secretase expression. e, f Knock-down of these genes via lentivirus-mediated shRNA expression in primary neurons decreases delta-secretase expression. The primary cultures were treated by OGD 2 h/reperfusion for 24 h. Neurons were collected and lysed. The mRNA and protein levels of AEP were detected by real-time PCR (c, e) or western blotting analysis (d, f). HSF1, C/EBPα, and C/EBPβ’s expression levels were detected by western blotting as well (d, f). Western blot data in a, b, d, and f are representative of three independent experiments. Real-time PCR data in a–c, e represent mean ± s.e.m. of three independent experiments (a–c, e: *P < 0.05, Student’s t-test)
