Fig. 3

Knockdown of Notch1 reduces inflammatory cytokine production in activated-astrocytes by IL-9. a The 3 recombinant lentivirus plasmids of Notch1-shRNA targeting astrocytes were transduced into mouse primary astrocytes using the Neon™ electron transfection system (MPK5000) for 48 h. The expression of Notch1 protein in astrocytes was evaluated by Western blot assay. b Primary mouse astrocytes were infected with recombinant lentivirus (LV-Notch1-shRNA, LV-ctrl) for 72 h, and then incubated in a serum-free medium overnight followed by treating with IL-9 for 6 h. Western blot analysis for the proteins of Notch1 pathway. c The mRNA levels of IL-6, TNF-α, and IP-10 in astrocytes were measured by real-time PCR. d The secretion levels of IL-6, TNF-α, and IP-10 in the supernatant of astrocytes were detected by CBA assay. **p < 0.01 and ***p < 0.001 versus DMEM group; ^#p < 0.05 and ^##p < 0.01 and ^###p < 0.001 versus LV-ctrl+IL-9 group (one-way ANOVA, the two-tailed Student’s t test). The data are from three independent experiments and represented as mean ± SEM