Fig 2: Hypertrophy and Increased Axonal Density in TSC iPSC-Derived Neuron-OL Co-cultures(A–D) Immunocytochemistry of co-cultures, showing SMI312 and DAPI expression in CN + CO (A), CN + PO (B), PN + CO (C), and PN + PO (D) groups.(E–H) Nissl soma staining in co-cultures of CN + CO (E), CN + PO (F), PN + CO (G), and PN + PO (H) groups.(I and J) Total axonal pixel density (I) and average soma size (J) per cell in co-cultures measured at day 28 of co-culturing. Mean per group (means ± SEM) was obtained in total from n = 4–9 independent experiments including C1-, C3-, P1B-, and P2-derived neurons and OLs. Scale bars, 25 µm. Asterisks in (I) and (J) indicate statistically significant differences found in one-way ANOVA and Tukey's post hoc test, *p < 0.05, **p < 0.005, only the most important differences are represented in the graph.See also Figures S4 and S5.

Fig 3: Loss of TSC1 or TSC2 reduces the expression of miR-125b-5p. (A) Tsc2+/+ and Tsc2-/- MEFs. (B) pLXIN (V) or pLXIN-hTSC2 retroviruses infected Tsc2-/- MEFs. (C) Tsc1+/+ and Tsc1-/- MEFs. (D) Kidney cystadenomas (Tumor) and the adjacent normal tissues (Normal) from Tsc2+/- mice (n=3). The expression of miR-125b-5p was detected by qRT-PCR. Error bars indicate mean ± SD of triplicate samples. **P<0.01; ***P<0.001.

Fig 4: mTORC1 upregulates STAT3 and downregulates miR-125b-5p expression. (A, B) Tsc2-/- MEFs (A) or Tsc1-/- MEFs (B) were treated with DMSO or with 50 nM rapamycin (Rapa) for 24 h. (C, E) Tsc2-/- MEFs (C) or Tsc1-/- MEFs (E) were infected with lentiviruses harboring vectors encoding Raptor shRNA (shRaptor) or the control shRNA (shSc). (D, F) The shRNA that silences Rictor (shRictor) or a control shRNA (shSc) were stably expressed in Tsc2-/- MEFs (D) or Tsc1-/- MEFs (F). (G) Pten+/+ and Pten-/- MEFs. (H) Tsc2+/+ MEFs were transduced with the retroviruses for myrAKT1 in pLXIN or its control vector pLXIN (V). (A-H) Cell lysates were subjected to western blot analysis using the indicated antibodies (left panels). The level of miR-125b-5p was detected by qRT-PCR (right panels). Error bars indicate mean ± SD of triplicate samples. **P<0.01; ***P<0.001. n.s indicates no significant difference.

Fig 5: Differential Proliferation and Maturation of TSC OLs in Co-cultures(A) Schematic of neuron-OL co-culture protocol in the Std and Std + Rapa conditions.(B–E) Immunocytochemical images of co-cultures showing OL precursor marker OLIG2- and OL maturity marker MBP-positive cells in the CN + CO (B), CN + PO (C), PN + CO (D), and PN + PO (E) groups.(F and G) Ratio of MBP/OLIG2- (F) and OLIG2/DAPI-positive cells (G) in the co-cultures at day 28. Mean per group (means ± SEM) was obtained in total from n = 4–9 independent experiments including C1-, C3-, P1B-, and P2-derived neurons and OLs.Scale bars, 25 µm. Asterisks in (G) indicate statistically significant differences found in one-way ANOVA and Tukey's post hoc test. While several differences were found using Tukey's post hoc only important differences are represented in the graph, *p < 0.05, **p < 0.005, ***p < 0.001, ****p < 0.0001. See also Figures S3–S5.