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).