2.2.2. (f) Rv2615c-induced apoptotic cell death involves
activation of Caspase 3 and 7
Classical intrinsic apoptosis includes the activation of Caspase 3 and
Caspase 7 as the ultimate executioner caspases following
APAF1-Caspase9-apoptosome formation. As a result, we investigated the
activation of Caspase 3 and Caspase 7 during Rv2615c-mediated cell
death. Rv2615c protein leads to activation of Caspase 3 and 7 at all the
time points shown by significant increase in the percentage of Caspase 3
and 7 positive cells (Fig 2c). Rv2615c-stimulation led to a significant
1.5-fold increase in Caspase 3 and 7 activations than unstimulated
cells. Staurosporine and LPS included as positive controls in the study
led to approximately 1.6-fold increase in percentage of Caspase 3 and 7
positive cells than the unstimulated cells at 48h. The percentage of
Caspase 3 and 7 positive cells was decreased by approximately 1.5 folds
when pre-treated with pan caspase inhibitor Z-VAD-fmk in cells
stimulated with Rv2615c protein and controls at 24h (Fig 2d).
Additionally, the percentage of AnnexinV/AnnexinV-PI dual positive cells
stimulated with Rv2615c protein decreased by 1.3-fold when pre-treated
with pan Caspase inhibitor Z-VAD-fmk at 24h (Fig 2e). These results
depicted that the selected Rv2615c protein induces Caspase 3 and 7
dependent apoptotic cell death in THP-1 macrophages.