FIGURE 6.

Knockdown of FoxO3a by shRNA represses up-regulation of endogenous Puma in neuronal cells subjected to Aβ treatment. A, cortical neurons were transfected with shFoxO3a or shRand and maintained for 48 h and then treated with Aβ (1.5 μm) for 8 h, after which they were immunostained with antibodies against Puma (red). Images were taken under a ×63 objective. B, percentage of stained cells pertains to the proportions of transfected cells (green) that show Puma staining either greater than that of non-treated control neurons (High) or equal to or less than that of non-treated neurons (Low). Data represent the mean ± S.E. (error bars) of three experiments. The number of cells evaluated per culture was ∼50. *, p < 0.01. C, graphical representation of corrected total cell fluorescence of Puma in neurons transfected with shRand or shFoxO3a following Aβ exposure. Difference in intensity of Puma staining was quantified by ImageJ as described under “Experimental Procedures.” Data represent mean ± S.E. of 60 different cells from three independent experiments. *, p < 0.03. D, PC12 cells were transfected with shFoxO3a or shRand and primed as described under “Experimental Procedures,” and then the down-regulation of endogenous FoxO3a was analyzed by Western blotting with anti-FoxO3a antibody. E, graphical representation of densitometric analysis of -fold change of FoxO3a level upon transfection with shFoxO3a or shRand in the presence or absence of Aβ (5 μm). Data represent mean ± S.D. (error bars) of two independent experiments. *, p < 0.05. F, PC12 cells were transfected with shFoxO3a or shRand and primed, and then the down-regulation of endogenous Puma was analyzed by Western blotting with anti-Puma antibody. G, graphical representation of densitometric analysis of -fold change of Puma level upon transfection with shFoxO3a or shRand in the presence or absence of Aβ (5 μm). Data represent mean ± S.D. of two independent experiments; *, p < 0.05.