Figure 5

Comparison of phagocytosis and intracellular survival of MS and MS-G in normal and in PKC-α deficient THP-1 cells. (A) THP-1 cells were incubated in the presence of 30 nM PMA for 24 h. Cells were then transfected either with SiRNA targeting PKC-α (ΔA) or scrambled SiRNA (S) and after 24 h were infected with MS or MS-G (MOI = 1:10) for 2 h, washed and remaining extracellular bacilli were killed by amikacin treatment for 1 h, again washed and internalized bacteria were released by lysis of macrophages with 0.05% SDS and plated then cfu were counted, (S/MS) phagocytosis of MS by normal THP-1 cells, (ΔA/MS) phagocytosis of MS by PKC-α deficient THP-1 cells, (S/MS-G) phagocytosis of MS-G by normal THP-1 cells, (ΔA/MS-G) phagocytosis of MS-G by PKC-α deficient THP-1 cells. 'T' test was performed for statistical analysis of data. (B) % survival of MS and MS-G in normal and PKC-α deficient THP-1 cells. Because, phagocytosis of MS and MS-G were different in control and in PKC-α deficient cells, cfu at 0 h was considered 100% and survival of MS is presented as percentage of the initial cfu. (C) At each time point of experiment, level of PKC-α in cells transfected either with SiRNA targeting PKC-α or scrambled SiRNA was also determined by immunoblotting, to confirm the levels of PKC-α throughout the experiment. Data are means ± standard deviations from three independent experiments each performed in 4 replicates. (*** = p < 0.0001).