Figure 6
BH4-Bcl-2, but not BH4-Bcl-Xl, protects against CytC- and STS-induced apoptosis via its interaction with the IP3R. (a–c) Loading of CytC (10 μM) in C6 glioma cells provoked a dramatic increase in the number of caspase-positive cells and cells with a fragmented nucleus (both quantified 25 min later) compared with cells loaded with buffer only ($ in (a) indicates that CytC significantly increases the apoptotic index compared with the control loading condition lacking CytC; the significance indication for CytC versus control was omitted from the other panels for clarity reasons). All results were obtained from four to eight independent experiments and are plotted as mean±S.E.M. Statistically significant differences are indicated as in Figure 5. (a) Co-loading of the IP3R peptide corresponding to the CytC-binding site (IP3RCYT 50 μM) together with CytC significantly (*) reduced the apoptotic index, indicating that CytC-induced apoptosis was amplified by its action on the IP3R. (b) BH4-Bcl-2, BH4-Bcl-2 K/D, BH4-Bcl-Xl and BH4-Bcl-Xl D/K (20 μM) significantly (*) protected against CytC-induced apoptosis in both assays, but not BH4-Bcl-2SCR. However, BH4-Bcl-2 was significantly ($) more potent than BH4-Bcl-Xl. Furthermore, Bcl-2 is significantly more potent than Bcl-2 K/D (+), whereas Bcl-Xl D/K is significantly more potent than BH4-Bcl-Xl (#). (c) BH4-Bcl-2-mediated protection against CytC-induced apoptosis was alleviated by co-loading with 20 μM IDP, whereas the antiapoptotic activity of BH4-Bcl-Xl was not affected by IDP. Thus, BH4-Bcl-2, BH4-Bcl-Xl and BH4-Bcl-Xl+IDP significantly (*) reduced CytC-induced apoptosis, but not BH4-Bcl-2+IDP. Furthermore, BH4-Bcl-2+IDP is significantly ($) different from BH4-Bcl-2. (d) Characterization of the STS (2 μM)-induced cell death profile by analyzing the percentage of cells staining positive for propidium iodide (PI+), caspase activation (Caspase) and both PI and caspase activation (PI+ and Caspase) as a function of time. (e) Similar approach as in (c) but with STS as an apoptotic trigger (applied in the medium for 6 h at 2 μM). (f) Similar approach as in (a–c), but with PAC-1, a small molecular activator of caspase-3, as an apoptotic trigger (applied in the medium for 6 h at 150 μM). (g) Effect of combining BH4-Bcl-2 and BH4-Bcl-Xl (both 20 μM) on CytC-induced apoptosis. *All conditions significantly reduced CytC-induced apoptosis. $The combination of BH4-Bcl-2 and BH4-Bcl-Xl is significantly different from BH4-Bcl-Xl, but not from BH4-Bcl-2. These results indicate no additive effect by combining BH4-Bcl-2 with BH4-Bcl-Xl. (h) Effect of BH4-Bcl-2 and BH4-Bcl-Xl on CytC-induced apoptosis in the presence of IP3RCYT peptide. *All conditions significantly reduced CytC-induced apoptosis. However, the protective effects of both BH4-Bcl-2 and BH4-Bcl-Xl were ablated in the presence of IP3RCYT peptide (50 μM) and thus did not significantly differ from the vehicle-treated condition
