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. 2014 Nov;141(21):4076–4086. doi: 10.1242/dev.108282

Fig. 7.

Fig. 7.

mTOR activity is regulated by GSK3 in the developing neural progenitors. (A) GSK3 elimination increases mTOR activity in the developing brain. mTOR activity was mostly localized in the ventricular zone in E13.5 control brains (Gsk3α+/−; Gsk3βloxP/+; Nestin-cre) as marked by phopho-S6K and phospho-4EBP1. In Gsk3α−/−; Gsk3βloxP/loxP; Nestin-cre brains, however, mTOR activity was expanded throughout the cerebral cortex. Scale bar: 25 μm. (B) Top: western blots at E13.5 showed that the levels of phospho-S6, phospho-S6K and phospho-4EBP1 were increased in GSK3-deficient brain tissues compared with the control. This indicates that GSK3 regulates mTOR activity in the developing brain. Control is Gsk3α+/−; Gsk3βloxP/+; Nestin-cre; knockout is Gsk3α−/−; Gsk3βloxP/loxP; Nestin-cre. Bottom: cortical progenitors from E13.5 mice were cultured in the presence or absence of GSK3 inhibitor lithium and then western blotting was performed using the cell lysates. Pharmacological inhibition of GSK3 increased mTOR activity and confirmed the in vivo results. (C) Quantification of western blots in B. Data are mean±s.e.m. *P<0.05. (D) Overexpression of GSK3 suppresses mTOR activity in cortical progenitors. Plasmids encoding a constitutively active GSK3β-GFP (ca-GSK3β-GFP) or GFP (control) were electroporated into E13.5 cortical neural progenitors. Cells were then cultured for 2 days and immunostained with phospho-4EBP1 antibody. Electroporated cells were identified by GFP expression (arrows). Cells expressing ca-GSK3β showed markedly reduced phospho-4EBP1 levels, whereas cells with control GFP expression had no effect. Scale bar: 20 μm. (E) The fluorescence intensities of phospho-4EBP1, as indicated in D, were quantified by ImageJ and are shown as relative changes versus control. *P<0.05. Data are mean±s.e.m. (F) Western blotting was performed using the lysates of the progenitors that were transfected with ca-GSK3β. The levels of phospho-S6 were reduced in cells expressing active GSK3β. (G) Quantification of phospho-S6 levels shown in F. *P<0.05. Data are mean±s.e.m.