Fig. 2.

MECP2 duplication promotes neurogenesis of cultured NPCs and ADAM10 is a critical down-stream molecule of MeCP2 in regulating NPCs differentiation. Mouse primary NPCs isolated from C57BL/6 mouse E12.5 embryonic cortex were infected with MeCP2 expressing lentivirus and cultured for 72 h. Cells were subjected for immunofluorescent staining (a) and cell lysates were subjected to western blotting (b–d). a Immunofluorescent staining for GFAP, MAP2, and NESTIN were performed on NPCs (N = 9). The MeCP2 and control viruses contain EGFP and showed here as green. The staining for MAP2, GFAP, and NESTIN are labeled with pseudo-colored red. Representative images are shown on the left and the percentage of Nestin⁺, MAP2⁺ and GFAP⁺ cells within EGFP⁺ cells are shown on the right panels respectively. Scale bar is 50 μm. b Western blot analysis for MAP2, GFAP, HA labeled MeCP2 and total MeCP2 (N = 9). GAPDH was used as loading control. Representative blot is shown on the left, and statistical analysis for MAP2, GFAP and MeCP2 levels are shown in the right panels, respectively. c Western blot analysis for components of NOTCH pathway, including NOTCH1, NICD, ADAM10, ADAM17, JAG1, and DLL1 (N ≥ 5). GAPDH was used as a loading control. Representative blots are shown on the left and statistical analyses for each component were presented on the right panels. d Overexpression of ADAM10 together with WT MeCP2 could reverse the differentiation effects of MeCP2 in cultured NPCs. HA tagged MeCP2 were transfected with either control vector or ADAM10 expressing plasmid (N ≥ 5). Seventy-two hours later, NPCs lysates were subjected to Western blot analysis for MAP2, GFAP, ADAM10, and HA. GAPDH was used as a loading control. Representative blots are shown on the left panel and statistical analyses were presented on the right panel, respectively