Figure 6.

Rapamycin-induced quiescence of cultured neurosphere cells is reversible. Primary neurospheres were dissociated and plated in differentiation conditions for either (1) 5 d (supplemented with DMSO or rapamycin), (2) 9 d (5 d with rapamycin plus 4 d of rapamycin withdrawal), or (3) 9 d (DMSO or rapamycin for the entire period) as indicated. A, Immunocytochemistry for βIII-tubulin+ neurons and GFAP+ astrocytes; note the reappearance of neurons and GFAP immunoreactivity following rapamycin withdrawal. B, Quantification shows that βIII-tubulin+ neuron number increases following rapamycin withdrawal. C, Western blotting of lysates from a parallel experiment; note the recovery in P-S6-RP and P-4EBP1 levels following rapamycin withdrawal and the associated increases in βIII-tubulin, GFAP, and CNPase levels. Five micrograms of protein were loaded per lane, and all bands for each probe were obtained from the same blot. *p < 0.05; **p < 0.01. Scale bar, 25 μm.