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.