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.