Figure 6

Involvement of Bcl-2 proteins in the cellular response. (A) Infection with Sindbis results in a shift in anti-apoptotic Bcl-2 family heterodimer composition. Cell lysates were collected from SV (+) or mock (-) infected MOSEC cells 20 h.p.i. and subjected to mitochondrial isolation. Fractionated lysates were immunoprecipitated with antibodies specific to Bad (cytoplasmic fraction), Bcl-xl and Mcl-1 (mitochondrial fraction). (B) Bax remains in the cytoplasm following infection with Sindbis vector. MOSEC cells were infected with SV-Luc or treated with staurosporine, as positive control. At 20 h.p.i. samples were subjected to immunofluorescence staining for Bax. Scale bars indicate 10 μm. (C) Knockdown of proteins using targeted siRNA is significant. MOSEC or Pan02 cells were transfected with indicated siRNAs. Lysates were collected 24 h.p.i. and subjected to western blotting for indicated proteins. (D) Translation is still inhibited in cells after protein knockdown. MOSEC cells transfected with siRNA directed against indicated proteins and infected with SV or mock infected were subjected to ³⁵S methionine labeling 24 h.p.i. (E) Ablation of expression of certain BH3 only proteins can partially protect cells from Sindbis vector infection. MOSEC or Pan02 cells were treated with indicated siRNAs (or siGLO control) and infected with Sindbis vector. Cell viability was assessed 24 h.p.i. Data in E represents the SEM (error bars) of three experiments. Cell viability for each sample was compared to the infected control and corrected for percentage of infection. Statistical significance was calculated by a two-tailed student t-test (* P < 0.05, ** P < 0.005).