Journal: Mucosal immunology

Article Title: Bcl-x L mediates RIPK3-dependent necrosis in M. tuberculosis -infected macrophages

doi: 10.1038/mi.2017.12

Figure Lengend Snippet: ( A ) Bcl-x L is required for pro-caspase 8 and RIPK3 accumulation on the mitochondria of H37Rv infected Mφ. Human Mφ were transfected with Bcl-x L or scrambled control (Scr) siRNA and then infected with H37Rv (MOI 10). Equal amounts of purified mitochondria from non-infected and H37Rv-infected Mφ were subjected to Western blot analysis to determine the levels of pro-caspase 8 and RIPK3. ( B ) Caspase inhibition of H37Ra infected Mφ enables RIPK3/Bcl-x L accumulation on mitochondria. After treatment with the caspase inhibitor z-IETD pro-caspase 8, RIPK3 and Bcl-x L accumulates on the mitochondria of H37Ra infected Mφ. Mitochondria were isolated from H37Rv or H37Ra-infected Mφ treated with or without z-IETD (10 μmol) and subjected to Western blot analysis using anti caspase 8, anti-RIPK3 and anti-Bcl-x L ab. Equal amounts of the proteins were subjected to Western blot analysis for the evaluation of RIPK3 and Bcl-x L levels. ( C ) Mφ treated with Bcl-x L 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 Mφ 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-x L . Mitochondria and cytosolic fraction of H37Rv-infected Mφ treated with RIPK3 or Scr siRNA were subjected to Western blot analysis and the levels of RIPK3, pro-caspase 8 and Bcl-x L were evaluated. (F ) Silencing of the RIPK3 gene activates apoptosis executor caspase 3 in H37Rv infected Mφ. Mφ 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 Mφ. Mitochondria were isolated from H37Ra (right panel) or H37Rv (left panel) -infected Mφ and the kinetics of BID processing and tBID accumulation were assessed by Western blotting. ( H ) Silencing of the BAX gene in H37Ra-infected Mφ blocks caspase 3 activation, a marker for apoptosis. Mφ 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.

Article Snippet: Human BAX-specific siRNA (#6321) was from Cell Signaling Technology.

Techniques: Infection, Transfection, Purification, Western Blot, Inhibition, Isolation, Staining, Activation Assay, Marker

Journal: PLoS ONE

Article Title: Andrographolide Analogue Induces Apoptosis and Autophagy Mediated Cell Death in U937 Cells by Inhibition of PI3K/Akt/mTOR Pathway

doi: 10.1371/journal.pone.0139657

Figure Lengend Snippet: (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 IC 50 (mean ± SEM) from three independent experiments (***p<0.001, compared to only AG–4 treated cells).

Article Snippet: The antibodies against Beclin 1 (3495), LC3 (3868), Atg 3 (3415), Atg 5 (8540), Atg 7 (8558), Atg 12 (4180), mTOR (2983), p-mTOR Ser2481 (2974), Raptor (2280), Rictor (2114), GβL (3274), Akt (4691), p-Akt Ser473 (4060), p-c-Raf Ser259 (9421), p-GSK–3β Ser9 (5558), p-PDK1 Ser241 (3438), PI3K(p85) (4257), p-PI3K p85(Tyr458)/p55(Tyr199) (4228), LY294002 (9901) and siRNA against Atg 5 (6345), Bax (6321), Akt (6211), mTOR (6381) and siControl (6568) were procured from Cell Signaling Technology (Inc. Beverly, MA, USA).

Techniques: Transfection, Western Blot, Inhibition

Journal: PLoS ONE

Article Title: Andrographolide Analogue Induces Apoptosis and Autophagy Mediated Cell Death in U937 Cells by Inhibition of PI3K/Akt/mTOR Pathway

doi: 10.1371/journal.pone.0139657

Figure Lengend Snippet: (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 IC 50 (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.

Article Snippet: The antibodies against Beclin 1 (3495), LC3 (3868), Atg 3 (3415), Atg 5 (8540), Atg 7 (8558), Atg 12 (4180), mTOR (2983), p-mTOR Ser2481 (2974), Raptor (2280), Rictor (2114), GβL (3274), Akt (4691), p-Akt Ser473 (4060), p-c-Raf Ser259 (9421), p-GSK–3β Ser9 (5558), p-PDK1 Ser241 (3438), PI3K(p85) (4257), p-PI3K p85(Tyr458)/p55(Tyr199) (4228), LY294002 (9901) and siRNA against Atg 5 (6345), Bax (6321), Akt (6211), mTOR (6381) and siControl (6568) were procured from Cell Signaling Technology (Inc. Beverly, MA, USA).

Techniques: Western Blot, Expressing, Transfection, Staining