Figure 5.

Metformin-induced aPKC-CBP signaling represses Mgll expression to promote adult NPC neuronal differentiation. (A) qPCR for Mgll mRNA from differentiating WT and CbpS436A NPCs in the presence of metformin (met, 1 μM). *** P < 0.001 (n = 4 animals/group). (B) ChIP-qPCR analysis for CBP binding at Mgll promoter in differentiating WT and CbpS436A NPCs in the presence of metformin (1 μM), relative to corresponding WT NPC samples. * P < 0.05 (n = 3 animals/group). (C) Representative western blot images and quantitative analysis for Mgll protein levels in differentiating P2 WT and CbpS436A NPCs in the presence of metformin (1 μM) for 7 days, with GAPDH as a loading control and relative to one of WT NPC samples. ** P < 0.01 (n = 4 animals/group). (D-E) Immunofluorescent images (D) and quantitative analysis (E) of the percentage of βIII tubulin+ (red)/GFP+ (green) co-labeled neurons out of total GFP+ cells (left panel) and the percentage of βIII tubulin+(red)/ GFP- cells out of total GFP- cells (right panel) from differentiating P2 WT and CbpS436A NPCs following transfection with Scr or Mgll shRNAs, together with an eGFP plasmid, in the presence of metformin (1 μM) for 7 days. Data were analysed by two-way ANOVA, n = 3-4 animals/group, * P<0.05, ** P < 0.01; Scale bar: 100 μm. (F-G) Immunofluorescent images (F) and quantitative analysis (G) of the percentage of βIII tubulin+ neurons out of total live P2 WT and CbpS436A NPCs treated with 50% conditioned medium (CM) from differentiating P2 NPCs transfected with Scr or Mgll shRNAs. Data were analysed by two-way ANOVA, n = 3 animals/group, * P < 0.05, ** P < 0.01. Scale bar: 100 μm. Data are represented as mean ± SEM. See also Figure S4. Scr-sh: Scramble shRNA, Mgll-sh: Mgll shRNAs; CM-WT/Scr-sh: CM from WT NPCs transfected with Scr-sh; CM-WT/Mgll-sh: CM from WT NPCs transfected with Mgll-sh; CM-CbpS436A/Scr-sh: CM from CbpS436A NPCs transfected with Scr-sh; CM-CbpS436A/Mgll-sh: CM from CbpS436A NPCs transfected with Mgll-sh.