Figure 5.

siRNA-mediated knockdown of Beclin 1, but not of Atg5, abolishes starvation-induced sensitization of Ins(1,4,5)P₃R. (A) Western blot analysis of indicated proteins in HeLa cells treated with indicated siRNA duplexes. (B) Unidirectional ⁴⁵Ca²⁺-flux experiments in siRNA-treated permeabilized cells pretreated with (3 h) or without (0 h) HBSS. During the experiment cells were exposed to 0.7 µM Ins(1,4,5)P₃ (circles) or 10 µM A23187 (triangles). The arrows indicate the addition of Ins(1,4,5)P₃ or A23187. Representative traces are shown of fractional ⁴⁵Ca²⁺ release (%/2 min) as a function of time in cells treated with siRNA against BECN1 siBECN-1 1. (C) Unidirectional ⁴⁵Ca²⁺-flux experiments in control siRNA (siCtrl)-treated cells. (D) Unidirectional ⁴⁵Ca²⁺-flux experiments in siRNA-treated cells against ATG5 (siAtg5). (E) Quantitative analysis of the Ins(1,4,5)P₃-induced ⁴⁵Ca²⁺ release relative to the A23187-induced ⁴⁵Ca²⁺ release in cells pretreated with (3 h) or without (0 h) HBSS (n = 4). *p < 0.05; **p < 0.01; N.S. not significant (paired t-test).