Journal: Journal of Virology
Article Title: Zika Virus Infection Induced Apoptosis by Modulating the Recruitment and Activation of Proapoptotic Protein Bax
doi: 10.1128/JVI.01445-20
Figure Lengend Snippet: Impact of the downregulation of Bax, Bak, caspase-8, caspase-9, and caspase-10 expression on cell apoptosis induced by ZIKV infection. (A) Western blot of the specificity and effect of protein knockdown in siRNA-transfected cells. SY5Y cells were transfected with siRNA for 48 h and then cell lysates were prepared for immunoblot with antibody to Bak, Bax, beta-actin, caspase-8, caspase-9, and caspase-10. siControl: cells transfected with no targeting siRNA. siBax/Bak: cells transfected with both siBax and siBak. (B) ZIKV levels in the siRNA-transfected cells supernatants determined by immunofluorescence focus units assay analysis (FFU). SY5Y cells were transfected with siRNA targets to Bax, Bak, both Bax and Bak (Bax/Bak), caspase-8, caspase-9, and caspase-10. At 48 h posttransfection, cells were infected with ZIKV for another 12 h or 24 h. Cell supernatants were collected and subjected to FFU analysis with ZIKV-E antibody. siControl: cells transfected with no targeting siRNA. (C) Analysis of the impact of ZIKV infection on the viability of siRNA-transfected cells. Cells were transfected with siRNA as described above. At 48 h posttransfection, the cells were infected with ZIKV (MOI = 1) for 24 h and subjected to cell viability analysis by ELISA. Mock: uninfected cells. (D) Analysis of the impact of ZIKV infection on the release Cyt c in siRNA-transfected cells. The release of Cyt c in cytosolic fractions was determined as described in Materials and Methods. Staurosporine: siRNA-transfected cells treated with 30 nM staurosporine for 12 h. (E) Analysis of caspase-3 activity in cells after siRNA transfection and ZIKV infection. Caspase-3 activity was measured as described in Materials and Methods. Cyt c = cytosolic fractions of SY5Y cells incubated with Cyt c (10 μM). Data are from three independent experiments and the results are presented as mean ± SEM. ** indicates a significant difference at P values of 0.02 compared to the control; * indicates a significant difference at P values of 0.05 compared to the control.
Article Snippet: Caspase-3 activity assay kit, caspase-9 activity assay kit, and Annexin V-FITC were obtained from Biyotime, China. table ft1 table-wrap mode="anchored" t5 TABLE 1 caption a7 Antibodies/reagents Source Identifier Working dilutions Caspase-3 antibody CST #9662S 1:2,000 (WB) Cleaved-caspase-3 antibody CST #9661 1:500 (IF); 1:2,000 (WB) Caspase-8 antibody Abcam Ab108333 1:2,000 (WB) Caspase-9 antibody Abcam Ab32539 1:2,000 (WB) Caspase-10 antibody Abcam Ab32155 1:2,000 (WB) Bcl-X L antibody CST #2764 1:2,000 (WB) BSA antibody CST #66271 1:2,000 (WB) Cyt c antibody CST #11940 1:2,000 (WB) Flag antibody Santa Cruz Ab205606 1:4,000 (WB) Bak antibody Santa Cruz SC518110 1:500 (WB) Bak (Ab-1, clone TC-100) antibody Merck AM03 1:500 (F) Beta-actin antibody Santa Cruz SC8432 1:4,000 (WB) Cox IV antibody CST #4850 1:200 Bax (6A7) antibody Santa Cruz SC23959 1:200 (IP); 1:500 (F) HRP-conjugated anti-rabbit IgG Santa Cruz SC2004 1:1,000 (WB) FITC-conjugated anti-rabbit IgG Santa Cruz SC2012 1:1,000 (IF) FITC-conjugated anti-mouse IgG ZSGB-BIO ZF-0312 1:1,000 (IF) HRP-conjugated anti-mouse IgG Santa Cruz SC2010 1:1,000 (WB) zLEHD.Fmk Beyotime C1202 15 μM zDEVD.Fmk Beyotime C1206 25 μM Staurosporine Beyotime S1882 30 nM CICCP Sigma-Aldrich #555-60-2 10 μM Open in a separate window a WB: western blot; IF: immunofluorescence; F: Flow cytometry.
Techniques: Expressing, Infection, Western Blot, Transfection, Immunofluorescence, Enzyme-linked Immunosorbent Assay, Activity Assay, Incubation
