Fig 1: Model of RIPK3-dependent programmed necrosis and caspase 8-dependent apoptosis in Mf infected with MtbIn Mf infected with virulent Mtb RIPK3 and pro-caspase 8 present in the cytosol translocate to the mitochondria in presence of Bcl-xL and RIPK3 is activated RIPK3 enhances binding of HKII to VDAC on the outer mitochondrial membrane controlling mitochondrial glycolysis. At the same time activated RIPK3 triggers CypD-dependent formation of the mitochondrial permeability transition (MPT) pore via interaction between ANT and VDAC leading to leakage of the electron chain. Both mechanisms seem to be required for increasing ROS-dependent necrosis (right). In Mf infected with avirulent Mtb the RIPK3 and caspase 8 also translocate to the mitochondria but this step is quickly followed by activation of caspase 8 and degradation of RIPK3. Oligomerization of BAX and BAK, which in turn allows the release of pro-apoptotic molecules (e.g. cytochrome c) leads to apoptosis (left). The exact action mechanism of Bcl-xL function is unknown.
Fig 2: Bcl-xL enables pro-caspase 8 and RIPK3 accumulation on mitochondria of Mtb2 infected Mf(A) Bcl-xL is required for pro-caspase 8 and RIPK3 accumulation on the mitochondria of H37Rv infected Mf. Human Mf were transfected with Bcl-xL or scrambled control (Scr) siRNA and then infected with H37Rv (MOI 10). Equal amounts of purified mitochondria from non-infected and H37Rv-infected Mf were subjected to Western blot analysis to determine the levels of pro-caspase 8 and RIPK3. (B) Caspase inhibition of H37Ra infected Mf enables RIPK3/Bcl-xL accumulation on mitochondria. After treatment with the caspase inhibitor z-IETD pro-caspase 8, RIPK3 and Bcl-xL accumulates on the mitochondria of H37Ra infected Mf. Mitochondria were isolated from H37Rv or H37Ra-infected Mf treated with or without z-IETD (10 µmol) and subjected to Western blot analysis using anti caspase 8, anti-RIPK3 and anti-Bcl-xL ab. Equal amounts of the proteins were subjected to Western blot analysis for the evaluation of RIPK3 and Bcl-xL levels. (C) Mf treated with Bcl-xL or scrambled control siRNA (Scr) were infected with H37Rv (MOI 10) and cell death was assessed after 48 h using Live/Dead fixable dead cell stain kits (Invitrogen). (D) z-IETD blocks activation of the apoptotic caspase 9 and 3 essential for apoptosis induction. Equal amounts of cell lysates of Mf infected with H37Ra (pro-apoptotic strain) treated with or without z-IETD (10 µmol) were subjected to Western blot analysis for evaluating active caspase 3 and 9. (E) RIPK3 is required for accumulation of mitochondrial pro-caspase 8 and Bcl-xL. Mitochondria and cytosolic fraction of H37Rv-infected Mf treated with RIPK3 or Scr siRNA were subjected to Western blot analysis and the levels of RIPK3, pro-caspase 8 and Bcl-xL were evaluated. (F) Silencing of the RIPK3 gene activates apoptosis executor caspase 3 in H37Rv infected Mf. Mf treated with RIPK3 or Scr siRNA were infected with H37Rv. Cell lysate was collected and equal amounts subjected to Western blot analysis for cleaved caspase 3. (G) Bid processing in H37Ra and H37Rv-infected Mf. Mitochondria were isolated from H37Ra (right panel) or H37Rv (left panel) -infected Mf and the kinetics of BID processing and tBID accumulation were assessed by Western blotting. (H) Silencing of the BAX gene in H37Ra-infected Mf blocks caspase 3 activation, a marker for apoptosis. Mf treated with BAX or Scr siRNA were infected with H37Ra or H37Rv (MOI 10). Cell lysate was collected after 0 and 24 h and equal amounts subjected to Western blot analysis for pro-caspase 3 and cleaved caspase 3. VDAC and GAPDH were used as a loading control. Results are expressed as mean ± SD. Data were analyzed using one-way ANOVA. *, Values of P < 0.05 were considered to be significant. Data are representative of four independent experiments.
Fig 3: AG–4 induced apoptosis and autophagy are dependent on each other.(A, C) Effect of inhibitors on AG–4 induced Annexin V positivity and AVO formation. Cells were treated with Z-VAD-fmk (20 µM), 3-MA (10 mM, 4 h) or both Z-VAD-fmk and 3-MA or transfected with siAtg 5 or siBax followed by treatment with AG–4 (5.4 µM, 48 h). (A) Histograms depict percentage of apoptotic cells and are presented as the mean ± SEM from three independent experiments (***p<0.001, as compared with control; @@@p<0.001, as compared with only AG–4 treated cells). (C) Histograms depict percentage of cells with AVO and are presented as the mean ± SEM from three independent experiments (***p<0.001, as compared with control, @@@p<0.001, as compared with only AG–4 treated cells). (B, D) Effect of inhibitors on apoptotic and autophagic proteins. Cells were treated with Z-VAD-fmk (20 µM), 3-MA (10 mM, 4 h) or both Z-VAD-fmk and 3-MA or transfected with siBax or siAtg 5 followed by treatment with AG–4 (5.4 µM, 48 h). Whole cell lysates were prepared and subjected to immunoblot analysis using specific antibodies against Bax or Atg 5. Analysis was confirmed with three different sets of experiments. (E) Effect of simultaneous inhibition of apoptosis and autophagy on AG–4 induced cytotoxicity. Cells were treated with Z-VAD-fmk (20 µM) and 3-MA (10 mM, 4 h) followed by treatment with AG–4 (0–50 µM, 48 h). Cell viability was determined by MTS-PMS assay. Results are expressed as IC50 (mean ± SEM) from three independent experiments (***p<0.001, compared to only AG–4 treated cells).
Fig 4: Involvement of mitochondrial pathway in AG–4 induced apoptosis.(A) Loss of mitochondrial membrane potential. Cells were incubated with AG–4 (5.4 µM, 0–48 h) and loaded with JC–1 for flow cytometric analysis of mitochondria transmembrane potential. Data is a representative of three different experiments. (B) Effect on intracellular Ca2+. Cells preloaded with Fluo–4 AM were incubated with AG–4 (5.4 µM). The flow cytometric measurement of free cytosolic Ca2+ levels was seen as a fluorescent signal. Data are expressed as mean GMFC±SEM of three independent experiments (***p<0.001, as compared with control). (C) Altered expression levels of pro- and anti-apoptotic proteins. Whole cell extracts were made from control and AG–4 (5.4 µM, 0–48 h) treated cells and subjected to western blot analysis for Bcl–2, Bcl-xl, Bax, Bad. Analysis was confirmed with three different sets of extracts. ß-actin served as a loading control. Histogram shows the time dependent decrease in Bcl–2/Bax ratio (*p<0.05, ***p<0.001, as compared with control). (D) Contribution of Bax in AG–4 induced cytotoxicity. Cells were transfected with siBax for 48 h followed by treatment with AG–4 (0–50 µM, 48 h). Cell viability was determined by MTS-PMS assay. Results are expressed as IC50 (mean ± SEM) from three independent experiments (***p<0.001, compared to only AG–4 treated cells). (E) Contribution of Bax in AG–4 induced apoptosis. Cells were transfected with siBax for 48 h followed by treatment with AG–4 (0–50 µM, 48 h). The percentage of apoptotic cells was determined by Annexin V and propidium iodide dual staining. Results are expressed as mean ± SEM from three independent experiments (***p<0.001, compared to control cells; @@@ p<0.001 compared to only AG–4 treated cells). (F) Effect on cytochrome c release. Cytoplasmic and mitochondrial fractions were prepared from control and AG–4 treated (5.4 µM, 0–48 h) cells using mitochondria/cytosol fractionation kit as described in materials and methods and cytochrome c was analyzed by Western blotting. Data shown are from one of the three experiments.
Fig 5: AG–4 inhibits PI3K/Akt/mTOR pathway.(A) Control and AG–4 treated (5.4 µM, 0–48 h) cells were analyzed by western blot for phosphorylated and total PI3K expression. The results shown are representative of three experiments. Histograms represent densitometric analysis of relative phosphorylation levels of PI3K. (B) Control and AG–4 treated (5.4 µM, 0–48 h) cells were analyzed by western blot for Akt pathway proteins. Analysis was confirmed with three different sets of extracts. (C) Control and AG–4 treated (5.4 µM, 0–48 h) cells were analyzed by western blot for mTOR pathway proteins. The figure is a representative profile of three experiments. (D, E, G) Effect of inhibitors on AG–4 induced cytotoxicity, Annexin V positivity and AVO formation. Cells were treated with LY294002 (20 µM, 1 h); Rapamycin (20 nm, 1 h) or transfected with siAkt, simTOR followed by treatment with AG–4 (D) Cell viability was assessed by MTS-PMS assay. Data are presented as IC50 (mean ± SEM) from three independent experiments (*p<0.05, **p<0.01, as compared with only AG–4 treated cells). (E) Histograms depict percentage of apoptotic cells and are presented as the mean ± SEM from three independent experiments (***p<0.001, as compared with control; @@p<0.01 & @p<0.05, as compared with only AG–4 treated cells). (G) They were then analysed for AVO by AO staining. Histograms depict percentage of cells with AVO and are presented as the mean ± SEM from three independent experiments (***p<0.001, as compared with control; @@@p<0.001, @@p<0.01 & @p<0.05, as compared with only AG–4 treated cells). (F, H) Effect of inhibitors on apoptotic and autophagic proteins. Cells were treated with LY294002 (20 µM, 1 h), Rapamycin (20 nm, 1 h) or transfected with siAkt, simTOR followed by treatment with AG–4 (5.4 µM, 0–48 h). Whole cell lysates were prepared and subjected to immunoblot analysis using specific antibodies against Bax or Atg 5. Analysis was confirmed with three different sets of experiments.
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