Figure 5.

Akt ‘recovers' its protective function on Ca²⁺ cell death in SH-SY 5Y-expressing IP₃R type III. (a) Immunoblotting showed the effective presence of isoform III of IP₃R in a different kind of SH-SY 5Y (termed SH 2). COS7 cells were used as reference for the right molecular weight of IP₃R III. SH 1 stands for SH-SY 5Y type III-deficient cells, used in previous experiments. (b) ER Ca²⁺ measurements in mock-transfected and m/p-Akt-overexpressing SH 2 cells. Magnifications of first part of the release phase are reported (inset). (c) Mitochondrial Ca²⁺ homeostasis modulation after Akt activation in SH 2 cells. (d) SH 2 cells were loaded with the Ca²⁺ indicator Fura-2/AM and 340/380 nm ratio changes were measured. The experiment was performed similarly to that reported in Figure 3. (e) SH 2 cells were transfected with the indicated gene (control: mock transfected), harvested after 36 h, and the lysates were assayed for caspase 3 activity as described in Materials and Methods. Apoptosis was induced by treatment with 80 μM AA for 40 min. Traces are from a single representative experiment. (f) SH 2 cells were transfected and treated in the same manner of e, and the endogenous cleavages of PARP and caspase 3 were revealed through immunoblotting. (g) SH 2 cells were co-transfected in a 3 : 1 ratio with empty vector and mtGFP (upper panels) or m/p-Akt and mtGFP (lower panels). Mitochondrial morphology was analyzed using confocal microscopy, 36 h post-transfection, before and after treatment with 80 μM AA, for 20 min. The insets show a greater magnification of the mitochondrial network