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. 2015 Jul 1;10(7):e0131767. doi: 10.1371/journal.pone.0131767

Fig 6. Reciprocal regulation of oxidative burst and calcium routing regulate apoptosis in macrophages by Rv3416 and Nef.

Fig 6

For Panel A and B, PMA stimulated THP1 cells were treated with 50 μM H2O2 for 1h and stimulated with 1 μg/ml Pam3CSK4 along with 20 μg/ml Rv3416 and 15 μg/ml Nef for 24h. For Panel A, cells were stained with Annexin V-APC. Thin lines represent cells stimulated in the absence of H2O2 while the thick lines represent cells stimulated in the presence of H2O2. Data from one of three independent experiments are shown. Bar graphs adjacent to histogram in Panel A show relative MFIs of the histogram. For Panel B, PMA stimulated THP1 cells were stimulated with 1 μg/ml Pam3CSK4 along with 20 μg/ml Rv3416 or 15 μg/ml Nef or both for 24h and cytoplasmic extracts were probed for indicated molecules and analyzed by western blots. Numbers below the blots indicate the relative intensities of the bands. Data from one of three experiments are shown. For Panel C (PMA stimulated THP1 macrophages) and Panel E (Blood macrophages) were stimulated with 1 μg/ml Pam3CSK4 along with 20 μg/ml Rv3416 (dotted lines) or 15 μg/ml Nef (thin lines) or both (thick lines) for 2h for measuring oxidative burst. Thirty minutes prior to the incubation period, cells were loaded with 10μM DCFH-DA. At the end of the incubation period, cells were thoroughly washed with the culture medium and immediately analyzed for ROS levels by flow cytometry. Shaded histograms in Panel C and E represent cells stimulated with 1 μg/ml Pam3CSK4 only. Bar graphs adjacent to histograms in Panel C and Panel D show relative MFIs of the corresponding histograms. For Panel D (THP-1 macrophages) and Panel F (Blood macrophages) cells stimulated with 1 μg/ml Pam3CSK4 along with 20 μg/ml Rv3416 and 15 μg/ml Nef with or without EGTA or TMB-8 for 1h and ROS levels were measured. In Panel C P<0.004 for Pam vs Pam+Rv3416+Nef; In Panel D, P<0.005 for Pam+Rv3416+Nef vs Pam+Rv3416+Nef+TMB; P<0.005 for Pam+Rv3416+Nef vs Pam+Rv3416+Nef+EGTA. In Panel E, P<0.03 for Pam vs Pam+Rv3416+Nef; In Panel F, P<0.05 for Pam+Rv3416+Nef vs Pam+Rv3416+Nef+TMB; P<0.04 for Pam+Rv3416+Nef vs Pam+Rv3416+Nef+EGTA.