Figure 7.

Possible Molecular Regulation Involved in Astrocyte Reprogramming

(A) Expression of the indicated genes in astrocyte-derived NSCs at passage 2 analyzed by quantitative real-time PCR (in three independent neurospheres from reprogrammed astrocytes). (B) Immunostaining analysis showing SOX2 expression in iNSCs (passage 2) derived from astrocytes induced by the indicated conditions depicting the enhancing effect of Shh on NSC generation and characteristics. Scale bars, 150 μm. (C) Quantitative real-time PCR and western blot analysis of Ptch, GLI2, and CyclinD1 mRNA and protein levels in astrocytes induced by the indicated conditions. (D) Western blot analysis of Smurf2, Nolz1, CDk2, PI3K, and phosphorylated PI3K expression in astrocytes under the indicated conditions after 10 days. (E) The analyses of astrocyte reprogramming mediated by Oct4/Shh after suppression of PI3K/Cdk2/Smurf2 signaling. β-Actin served as a loading control. Notably, no changes in the expression of the aforementioned molecules were found in control astrocytes or mock-transduced astrocytes. mRNA expression was normalized to that of GAPDH mRNA. The results were obtained from three independent experiments. All data are reported as the means ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001 versus the corresponding controls. GDC-0941 is a PI3K inhibitor, SU9516 a CDk2 inhibitor, and USF2 a Smurf2 inhibitor. CDk2, cyclin-dependent kinase 2; CTX, cortex; GLI2, GLI family zinc-finger 2; Nolz1, Mus musculus zinc-finger protein Nolz1; Ptch, patched homolog; Smurf2, Smad ubiquitin regulatory factor 2; Transd, transduction.