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Fig. 3. Requirement of STAT3 for maintenance of FGF2-sensitive NPCs. (A) NPC cultures prepared from E12.5 mouse neocortex were incubated in the absence of FGF2 for the indicated times, after which cell lysates were subjected to immunoblot analysis with antibodies to STAT3, to the Tyr⁷⁰⁵-phosphorylated form of STAT3 (Anti-phospho STAT3) and to GAPDH (loading control). Reduction of STAT3 phosphorylation by FGF2 deprivation suggests FGF2-dependent phosphorylation of STAT3, although it may also reflect the reduction of sphere-forming NPCs by FGF2 deprivation. (B) Neuroepithelial cells prepared from the neocortex of E12.5 STAT3^flox/flox mice were infected with a retrovirus encoding Cre recombinase (Cre) or with a control retrovirus (Control). After culture in the presence of FGF2 (20 ng/ml) for the indicated times, the cells were lysed and subjected to immunoblot analysis. (C) Neuroepithelial cells from STAT3^flox/flox mice were infected and then assayed for the formation of primary neurospheres in the presence of FGF2 (20 ng/ml). ^*P<0.01. (D) Neuroepithelial cells prepared from the neocortex of E12.5 wild-type mice were infected with retroviruses encoding both STAT3-C and GFP (STAT3-C) or GFP alone (Control). They were subsequently assayed for the formation of secondary neurospheres. ^*P<0.01. (E-G,M) Neuroepithelial cells prepared from E12.5 wild-type mouse neocortex were infected and plated in the presence of FGF2 (20 ng/ml) for 2 days (E,F) or in the absence of FGF2 for 3 (G) or 4 (M) days. Then the cells were stained with Hoechst to identify apoptotic cells by counting cells exhibiting condensed and fragmented nuclei (F), or stained with anti-GFAP (G) or anti-ßIII-tubulin (TuJ1) antibody together with anti-GFP (M), or incorporated BrdU for 2 hours and stained with anti-BrdU and anti-GFP (E). ^*P<0.02. (H-L) Neuroepithelial cells prepared from E12.5 STAT3^flox/flox mouse neocortex were infected and plated in the presence of FGF2 (20 ng/ml) for 2 days (H,I) or a lower concentration of FGF2 (2 ng/ml) for 3 (J) or 4 (K,L) days. Then the cells were analyzed as in E-G,M. Scale bar: 80 µm (K). ^*P<0.01.

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