Figure 2. Western blot and graphs depicting activation of
Caspase9 and Caspase 3 and 7 respectively following protein stimulation
of THP-1 macrophages
Activated Caspase9, 3 and 7 estimation experiments were carried out on
THP-1 macrophages that had either been left unstimulated or stimulated
with control/Rv2615c protein. (a) Western blot image showing bands for
activated Caspase9. Proteins were transferred onto the PVDF membrane
after cell lysate was prepared and separated on SDS-PAGE post 24 hours
stimulation. The polyclonal anti-Caspase9 antibody was used to measure
the amounts of activated Caspase9, and an internal loading control,
GAPDH, was employed at a dilution of 1:1000. b) ImageJ software was used
to quantify the size of each western blot band. Results are shown as the
meanSEM values of three separate experiments and were examined by
showing the ratio of each band area with respect to unstimulated cells.
Student’s t test was performed where * depicts the comparison with
unstimulated cells for Caspase9 and GAPDH separately.
(*P<0.05, **P<0.01). c) Unstimulated/control/Rv2615c
protein stimulated THP-1 macrophages were incubated for different time
points (16h, 24h and 48h) and activation of Caspase 3 and 7 was
estimated which is a characteristic of intrinsic apoptosis.
Each western blot band was measured using ImageJ software. The ratio of
each band area with respect to unstimulated cells was investigated, and
the results are presented as them Mean±SEM values of three distinct
experiments. The comparison of Caspase9 and GAPDH individually with
unstimulated cells was shown using the Student’s t test, where *.
(*P<0.05, **P<0.01). c) THP-1 macrophages that were
left unstimulated or stimulated with control/Rv2615c protein were
incubated for different time points (16 hours, 24 hours, and 48 hours),
and the activation of caspase 3 and 7 was estimated. THP-1 cells were
also blocked with z-VAD-fmk Caspase inhibitor prior to stimulation and
evaluated for d) levels of Caspases 3 and 7 and e) expression of
AnnexinV/AnnexinV-PI dual positive cell population at 24h. Graphs were
plotted for different time points with % of positive cells on y axis
and control/Rv2615c on x axis. Statistical analysis was done with
Student’s t test and results are Mean ± SEM of three independent
experiments where * represents comparison with unstimulated cells and
*P<0.05, **P<0.01, ***P<0.001.