Fig. 1

Reduced functional maturation in the absence of TSC2. a Experimental protocol outline. For an experiment neural stem cells, generally maintained in self-renewing conditions (week −1), were subjected to a patterning step for seven days (week 0) before induction of neural differentiation (week 1–6). Samples were collected in weekly intervals for gene expression quantification by qPCR. After six weeks of differentiation, additional samples were collected for western blot, immunofluorescence, RNA-Seq, and ribosome profiling. b Relative quantification of expression levels of the indicated neural marker genes over the course of six weeks of differentiation in control and TSC2 deleted cell lines. Expression levels in control cells after six weeks of differentiation are normalized to 1. Error bars show standard error of the mean and asterisks indicate significant difference in a two-tailed Student’s t-test, *p < 0.05, **p < 0.01, ***p < 0.001. c Representative confocal images of 6-week-old neural cultures of control and TSC2 deleted cell lines stained for doublecortin (DCX) and GFAP. Confocal z-axis stacks were acquired and restructured. d, e Western blot analysis for the indicated proteins of neural cultures after six weeks of differentiation using independent derived control (C1, C2) and TSC2 deletion (T1, T2) cell lines in biological replicates (e.g. C1 and C1’). Densitometric analysis of GFAP protein and STAT3 phosphorylation levels is normalized to beta Actin and total STAT3 levels, respectively. Data are normalized to the level in control cell line C1 and error bars represent standard deviation. Significance is determined by Student’s two-tailed t-test, *p < 0.05, ***p < 0.001