DEV02419 Supplementary Material

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• Supplemental Figure 1 -

  Fig. S1. (A) Inhibition of FGF2-dependent NPC maintenance by simultaneous treatment with JAK2 and PI3K inhibitors. Neuroepithelial cells prepared from E12.5 mouse neocortex were dissociated and cultured in suspension with FGF2 (20 ng/ml) for 2 days and in the additional absence or presence of U0126 and LY294002 for 1 day. The resulting primary neurospheres were dissociated and cultured in suspension for 7 days with FGF2 (20 ng/ml) and EGF (20 ng/ml) in the absence of inhibitors. The number of secondary neurospheres was then counted. (B-D) Effects of inhibitors on NPC differentiation, proliferation and survival. Neuroepithelial cells prepared from E12.5 mouse neocortex were infected with a retrovirus encoding GFP (pMX-GFP) at a low titer (B) or a high titer (C,D), and plated in the presence of FGF2 (20 ng/ml) for 2 days and in the additional absence or presence of U0126, LY294002 or AG490 for 1 day. Then, the cells were stained with Hoechst to identify apoptotic cells by counting cells exhibiting condensed and fragmented nuclei (D); or they incorporated BrdU for 2 hours and were stained with anti-BrdU and anti-GFP (C); or after incubation for 3 days in the absence of FGF2, cells in each clone were stained with TuJ1 antibody and the number of clone containing only TuJ1⁺ cells was counted (neuron-only clone) (B). AG490 treatment promoted neuronal differentiation (B), slightly reduced proliferation (C) and had little effect on survival (D) of NPCs. *P<0.01.

• Supplemental Figure 2 -

  Fig. S2. STAT3 immunoreactivity was reduced by the conditional gene deletion of STAT3 gene. Vector encoding Cre recombinase plus GFP (pMX-Cre-GFP) was injected into the lateral ventricle of STAT3^flox/flox mice at E14.5 and were introduced into cells in the VZ by electroporation in utero. After 2 days, STAT3 was detected by immunohistochemistry (arrow), but not in GFP-positive cells (arrowhead), confirming that STAT3 protein was eliminated by STAT3 gene deletion. STAT3 staining indicate that STAT3 is localized mainly in the cytoplasm, and slightly in the nucleus. Scale bar: 5 μm.

• Supplemental Figure 3 -

  Fig. S3. Effects of STAT3 expression on NPC maintenance and survival. (A) Neuroepithelial cells from E12.5 wild-type mice infected with a retrovirus encoding STAT3-C (pMX-STAT3-C-GFP) or with a control retrovirus (pMX-GFP) were assayed for the formation of primary neurospheres in the presence of FGF2 (20 ng/ml). (B) Neuroepithelial cells from E12.5 STAT3^flox/flox mice were infected with a retrovirus encoding Cre-recombinase (pMX-Cre-GFP) or with a control retrovirus (pMX-GFP). They were subsequently assayed for the formation of secondary neurospheres. *P<0.01. (C) Neuroepithelial cells prepared from the neocortex of E12.5 STAT3^flox/flox mice were infected with a retrovirus encoding Cre recombinase (pMX-Cre-GFP) or with a control retrovirus (pMX-GFP). After formation of primary or secondary neurospheres, the cells were subjected to immunoblot analysis with anti-STAT3 and anti-GAPDH. The level of endogenous STAT3 was markedly increased in secondary neurospheres.

• Supplemental Figure 4 -

  Fig. S4. Requirement of STAT3 for inhibition of neurogenesis in vivo. (A) Vectors encoding GFP alone (pMX-GFP) or Cre recombinase plus GFP (pMX-Cre-GFP) were injected into the lateral ventricle of STAT3^flox/flox mice at E14.5, and were introduced into cells in the VZ by electroporation in utero. After 2 days, the fate of the GFP-positive cells was examined by immunohistochemistry with anti-GFP, anti-SOX2 and anti-MAP2, as indicated. Scale bar: 200 μm. Expression of Cre recombinase with GFP, but not that of GFP alone, resulted in ectopic expression of MAP2 and loss of SOX2 only in the area of vector introduction (expressing GFP). (B) pMX-GFP was introduced into cells at the VZ by in utero electroporation at E14.5, and cells expressing GFP were detected in the cortical plate 2 days after electroporation. GFP expression was detected in TuJ1⁺ cells (arrow). Scale bars: 40 μm.