Figure 8

Annexin V and Caspase 3 assays validate apoptosis by αDENV-ΔU143-ΔN Bax activation. A. Promotion of the initial stages of apoptosis by αDENV-ΔU143-ΔN Bax activation. Clonal Aedes albopictus C6/36 cells transformed with αDENV -U143-FL, αDENV -ΔU143-ΔN Bax or αDENV -U143-ΔN Bax were challenged with either one of the four DENV serotypes. 1x10⁶ cells were stained with FITC conjugated Annexin V at 48 hpi (with DENV) and analyzed in a 96-well microtiter plate format per the manufacturer’s instructions (Cayman Chemical Company). Uninfected clonal and wild type C6/36 cells were also assayed as an additional negative control. Assays were performed in triplicate. Error bars indicate standard deviation of three independent experiments. B. Caspase-3 activation confirms the induction of apoptosis by the activation of Dual Targeting Intron Constructs. Clonal Aedes albopictus C6/36 cells transformed with αDENV -U143-FL, αDENV-ΔU143-ΔN Bax or αDENV -U143-ΔN Bax constructs were challenged with either one of the four DENV serotypes (MOI 0.1). 1x10⁶ cells were assessed for caspase 3 activity, an indication of apoptosis at 4d.pi (for DENV), and analyzed for caspase activity in a 96-well microtiter plate format per the manufacturer’s instructions (see Methods). Uninfected clonal and wild type C6/36 cells were also assayed as an additional negative control. Assays were performed in triplicate. Error bars indicate standard deviation of three independent experiments. I = infected naïve C6/36 cells. U = uninfected naïve C6/36 cells. ΔU143 = the anti-DENV group I intron (αDENV-ΔU143) possessing the inactive deletion mutation of the trans-splicing domain.