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. 2021 Mar 25;95(8):e01445-20. doi: 10.1128/JVI.01445-20

FIG 4.

FIG 4

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.