Fig. 3. Survival of MLNSC in insulin correlates with insulin-induced Akt activation. Cell recovery and BrdU assays were performed on cultures of CD31^- CD35^- EGFP⁺ neurospheres grown in MB-media, MB-media minus insulin or MB-media plus 100 µM quercetin. (A) Sphere recovery was determined by counting the number of spheres daily and normalizing to the number of spheres grown in complete MB-media. Presented is the mean±s.d. for three experiments from distinct cellular isolations. (B) Proliferation is unaltered in BM-media. BrdU incorporation assays were conducted on neurospheres grown in MB-media, MB-media minus insulin and MB-media plus 100 µm quercetin. Little change in BrdU incorporation was observed whether BrdU was delivered for 4 (B) or 8 (C) hours. Presented is the mean±s.d. of three experiments normalized to neurospheres grown in MB-media containing insulin. (D) Insulin-induced survival correlates with the activation of Akt. Western analysis of neurospheres cultured in MB-media minus insulin (lane A), in MB-media plus 100 µM quercetin (lane B) and in MB-media (lanes C,D). (E) Caspase 3 is activated in neurospheres the absence of insulin signaling. Western analysis of procaspase 3 and activated caspase 3 in MB-media (lanes A,B) or MB-media plus 100 µM quercetin (lane C).