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. Author manuscript; available in PMC: 2019 Oct 1.
Published in final edited form as: Mol Cell Neurosci. 2018 Aug 23;92:149–163. doi: 10.1016/j.mcn.2018.08.004

Fig. 7.

Fig. 7.

PI3K and AKT inhibition, but not mTORC1, affects neuronal differentiation in NPCs. (A) Western blot analysis of AKT (Thr308) and S6 phosphorylation in control and TSC NPCs treated with 5 μM RAD001 or DMSO for 48 hours. (B, C) Quantification of Western blot data for the RAD001 treatment. Phospho-S6 (pS6) levels were significantly higher in TSC-DMSO compared to CTR-DMSO samples, but were strongly reduced by RAD001 treatment (B), whereas significantly lower levels of phospho-AKT (pAKT) were unaffected (C) (*p<0.05, ***p<0.001, ****p<0.0001, n=6 DMSO-treated cultures, n=3 RAD001-treated cultures). Significance was determined via one-way ANOVA with Tukey’s post-hoc analysis. (D, E) Western blot analysis of AKT (Thr308) and S6 phosphorylation in control NPCs treated with 1 μM MK2206, 10μM LY294002 or DMSO for 24 hours. (F) Quantification of the pAKT data. Both subject groups show significantly attenuated pAKT levels in MK2206-treated as well as LY294002-treated cultures compared to DMSO controls (*p<0.05, **p<0.01, ***p<0.001, n=3 cultures of 3 independent cell lines per subject). Significance was determined via one-way ANOVA with Dunnett’s multiple comparisons tests. (G) Representative confocal images of CTR #8 and TSC #6 cultures treated with DMSO, 5μM RAD001, 1μM MK2206 or 10μM LY294002, and differentiated for 7 DIV. Cultures were immunostained for the pan-neuronal marker HuC/D and counterstained with Hoechst. Scale bar = 50μm. (H, I) Automated high content analysis of treated culture images from both subject sets. The fraction of HuC/D+ cells was calculated from 3–6 experiments using 3 independent lines per subject, normalized to the internal control in each set (CTR+DMSO), and expressed as fold change. Significance was determined via one-way ANOVA with Dunnett’s multiple comparisons tests. (H) The fraction of HuC/D+ cells was significantly reduced in TSC #6+DMSO compared to CTR #8+DMSO cultures, and treatment with RAD001 did not rescue the defect. The fraction of HuC/D+ cells was similarly reduced in CTR #8+MK2206 and CTR #8+LY294002 cultures, but was not altered in CTR #8+RAD001 cultures. **p<0.01, ***p<0.001, n=4 independent cultures (RAD001 treatment), n=3 (MK2206 and LY294002 treatments), n=6 (DMSO). (I) The fraction of HuC/D+ cells was significantly reduced in MK2206- and LY294002-treated cultures compared to DMSO-treated CTR #5 cultures. *p<0.05, **p<0.01, n=3 independent experiment per treatment. Plots represent mean values ± SEM.