2.2.2. (b) Rv2615c protein did not induce LDH, a marker for the necrotic cell death
LDH release usually determines the cellular cytotoxicity in response to external stimulus; moreover, it also defines if the cells are undergoing necrotic cell death. 1% Triton-X, lysis buffer and a positive control was included as controls in study. Rv2615c-stimulation led release of LDH was observed to be at basal level, comparable to the unstimulated cells (Fig S3). Levels of LDH were significantly high in each of the positive controls included in the study at 16h, 24h and 48h. These observations indicate that the cell death induced by Rv2615c protein is not necrosis.
2.2.2. (c) Rv2615c-stimulation leads to flipping of phosphatidylserine on outer membrane and apoptosis of THP-1 macrophages
Phosphatidylserine [PS] is flipped from the inner to the outer leaflet of the plasma membrane as a result of biochemical alterations associated with the initiation of apoptotic pathway. Rv2615c-stimulation showed an increase in early apoptosis indicated by annexinV positive cells (Fig 1b, bottom right quadrant) plus late apoptosis indicated by annexinV/PI dual-positive cell population (Fig 1b, top right quadrant) in a time-dependent manner. At 16h, there was no significant increase in percentage of both the cell populations (early apoptotic annexinV positive; late apoptotic annexinV/PI positive cells) on stimulation with Rv2615c protein as compared to unstimulated control (Fig 1a). At 24h and 48h, Rv2615c-stimulation showed gradual and significant increase in percentage of dual positive cell populations (Mean±SEM in range of 37 to 49%) as compared to unstimulated cells (Mean±SEM in range of 27 to 35%) while the cell death was comparable to LPS stimulation (Mean±SEM range of 33 to 47%) at 48h. Since Staurosporine is a well-known cell death inducer; we observed maximum cell death (Mean±SEM value of 62%) till 48h (Fig 1a).