Fig. 8. Acute TANC2 knockdown in human neurons induces hyperactivity of mTORC1 and mTORC2.

a Schematic of the experimental design to test if TANC2 knockdown increases mTORC1/2 activity in human neural progenitor cell (NPC)-derived neurons (day 14), which are less likely to be fully matured neurons at this stage. Pan-NPCs infected with lentivirus particles for TANC2 knockdown were selected and subjected to neuronal maturation for 2 weeks before the analysis of mTORC1/2 signaling by immunoblot analysis. b, c Knockdown of TANC2 in human neurons leads to hyper-phosphorylation of S6 (S235/236), 4E-BP (T37/46), and GSK3β (S9), indicative of increased mTORC1 and mTORC2 hyperactivity. Note that the effects of the two knockdown constructs are not identical, in particular, for Akt (S473) phosphorylation, which was unaltered and increased by the #1 and #2 knockdown constructs, respectively. It could be because of distinct properties of the two constructs, such as differences in the strengths of target sequence binding, time courses of target gene knockdown, or compensatory cellular responses to adjust Akt activity. Human NPCs infected with two independent TANC2 knockdown lentivirus particles (#1 and #2) were differentiated into neurons and analyzed by immunoblot analyses. Data: mean ± SD. (n = 4 independent experiments; *P < 0.05, ***P < 0.001, ns not significant [compared to controls], one-way ANOVA with Bonferroni test). See Source Data 1 for raw data values and Supplementary Table 1 for statistical details.