Fig. 3.

Astrocyte-specific Dll4 inactivation induces downregulation of astrocyte reactivity under neuroinflammatory condition. (A-G) NA were transfected with a CONTROL siRNA (20µM) versus a DLL4 siRNA (20 µM) and treated with IL-1β 10ng/mL for 12 h. (A-C) DLL4 expression was quantified by (A) qRT-PCR (n = 6) and (B-C) WB. β-ACTIN (ACTB) was used as a reference. (B) Representative WB for DLL4 and ACTB protein expression are shown. (C) DLL4 protein expression was quantified. (n = 6). (D-E) NICD1, (D, F) VIM, (D, G) cleaved-CASP3 expression were quantified by WB. ACTB and CASP3 total were used as references. (n = 6) (H) Transcriptional RNA profiling of astrocyte lysates from EAE induced Dll4^ACKOC mice and control littermates was performed. A Heatmap of the differentially expressed genes involved in astrocyte reactivity [28] is shown. (I-K) VIM and GFAP protein expression were quantified by WB in spinal cord lysates from Freund adjuvant or EAE-induced Dll4^ACKOC mice and control littermates (at 18 days post induction). TUBULIN was used as a reference. (I) Representative WB for VIM, GFAP and TUBULIN are shown. (J-K) VIM and GFAP protein expression were quantified by WB. TUBULIN was used as a reference. (Dll4^ACKOP mice n = 6, WT n = 6) (Dll4^ACKOC mice n = 6, WT n = 6) (L-N) Spinal cord EAE lesions from Dll4^ACKOP mice, Dll4^ACKOC mice and littermate controls were harvested at 18 days post induction and immuno-stained with anti-GFAP (in green), anti-VIM (in red) and anti Podocalyxin (PODXL) (in grey) (to discriminate vascular from astrocytic VIM expression) antibodies. Nuclei were stained with DAPI (in blue). (L) Dll4^ACKOC mice versus control tissues are shown. (M) GFAP + and (N) VIM+/PODXL- areas were quantified (Dll4^ACKOP mice n = 6, WT n = 6) (Dll4^ACKOC mice n = 7, WT n = 7)). Statistical significance was determined by using a Mann-Whitney U test or Kruskal Wallis test followed by the Dunn’s multiple comparison test