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1.
Figure 2

Figure 2. Modulation of macrophage functions by soluble BAT3.. From: BAT3 Regulates Mycobacterium tuberculosis Protein ESAT-6-Mediated Apoptosis of Macrophages.

A. J774A.1 cells were first stimulated with IFN-γ (20 ng/ml) for 2 hours and then purified recombinant BAT3 was added into culture at different concentrations. Total nitrite levels in cell culture supernatants were determined after 18 hours using a colorimetric assay kit. **P<0.01 and ***P<0.001 as compared with IFN-γ only group. B. J774A.1 cells were first incubated with LPS (100 ng/ml) for 2 hours and then stimulated with BAT3 at different concentrations. The levels of IL-1β in cell culture supernatants were determined after 24 hours using ELISA kit from eBioscience Inc. **P<0.01 and ***P<0.001 as compared with LPS only group. C. J774A.1 cells were incubated with LPS (100 ng/ml) for 2 hours and then stimulated with BAT3 at different concentrations. The levels of IL-12p70 in cell culture supernatants were determined after 24 hours using ELISA kit from eBioscience Inc. ***P<0.001 as compared with LPS only group.

Ajay Grover, et al. PLoS One. 2012;7(7):e40836.
2.
Figure 6

Figure 6. Inhibition of caspase-3 and proteasome provides protection of BAT3 and BCL-2.. From: BAT3 Regulates Mycobacterium tuberculosis Protein ESAT-6-Mediated Apoptosis of Macrophages.

J774A.1 cells were pre-incubated with 85 µM zVAD-FMK or 10 µM proteasome inhibitor MG-132 or both for 4 hours and then stimulated with 5 µg/ml of ESAT-6 protein. Cytoplasmic extracts were collected at different time points. Total protein concentration of different fractions was determined and equal amounts of proteins (25 µg) were loaded on an SDS-PAGE gel. Following electrophoresis and western blotting, the blots were developed using a rabbit polyclonal antibody against BAT3 protein (Figure 6A) and a mouse monoclonal antibody against BCL-2 protein (Figure 6B). All experiments were done with 0.05% DMSO vehicle control and ESAT-6 alone as positive control (data not shown). The cytoplasmic marker GAPDH served as positive control for the western blots of cytoplasmic extracts.

Ajay Grover, et al. PLoS One. 2012;7(7):e40836.
3.
Figure 5

Figure 5. Interaction of BAT3 with BCL-2 and expression of BCL-2 in ESAT-6 stimulated J774A.1 cells.. From: BAT3 Regulates Mycobacterium tuberculosis Protein ESAT-6-Mediated Apoptosis of Macrophages.

A. J774A.1 cells were co-transfected with FLAG-tagged BAT3 plasmid and HIS-tagged BCL-2 plasmid or control vectors and incubated with ESAT-6. Cytoplasmic extracts of the cells were subjected to immunoprecipitation (IP) with antibodies against FLAG peptide or HIS tag, followed by western blotting with respective antibodies as indicated. Total 25 µg of each protein sample was loaded in 12% SDS-PAGE gel for the development of western blot. Upper panel: IP with anti-FLAG antibody and western blot with anti-HIS antibody. The molecular weight of BCL-2 was ∼25 kDa. Lower panel: IP with anti-HIS antibody and western blot with anti-FLAG antibody. The molecular weight of BAT3 was ∼122 kDa. Whole cell lysates of the J774A.1 cells expressing recombinant BAT3 and BCL-2 proteins served as positive control for the western blotting. B. Upper panel: J774A.1 cells were incubated with 5 µg/ml of ESAT-6 and cytoplasmic extracts were collected at different time points. Total protein concentration of different fractions was determined and equal amounts of proteins (25 µg) were loaded on an SDS-PAGE gel. Following electrophoresis and western blotting, the blot was developed using a mouse monoclonal antibody against BCL-2 protein. J774A.1 cells were either transfected with BAT3 plasmid (lower panel) or control vector (middle panel) for 72 hours, incubated with ESAT-6 and cytoplasmic extracts were collected at different time points. Western blots were developed for the BCL-2 protein as described earlier.

Ajay Grover, et al. PLoS One. 2012;7(7):e40836.
4.
Figure 4

Figure 4. Regulation of ESAT-6 induced apoptosis by BAT3.. From: BAT3 Regulates Mycobacterium tuberculosis Protein ESAT-6-Mediated Apoptosis of Macrophages.

A. BMDMs and J774A.1 cells were incubated with different concentrations of ESAT-6 for 24 hours. Cell culture supernatants were collected and subjected to colorimetric caspase-3 assay. Fold change in comparison to unstimulated controls was calculated and plotted in bar graph. *P<0.1 and **P<0.01 as compared with ESAT-6 (2 µg/ml) group. B. J774A.1 cells were transfected with BAT3 plasmid or control vector and then stimulated with 5 µg/ml of ESAT-6 protein for 24 hours. The cells were stained with PE labelled AnnexinV and 7-AAD and subjected to flow cytometry for determination of Annexin V positive and 7-AAD negative apoptotic cells. Percentages of apoptotic cells were plotted in a bar graph. J774A.1 cells treated with 5 µg/ml of Ag85B protein served as negative control for apoptosis. ***P<0.001 as compared with control plasmid+ESAT-6 group. C. J774A.1 cells were transfected with BAT3 siRNA or control siRNA or pre-incubated with 85 µM zVAD-FMK for 4 hours and then stimulated with 5 µg/ml of ESAT-6 protein for 24 hours. The cells were stained and subjected to flow cytometry to determine apoptosis as mentioned above in figure 4B. ***P<0.001 as compared with ESAT-6 only group. D. J774A.1 cells were transfected with control vector for 72 hours or BAT3 plasmid for time intervals ranging from 24 hours to 72 hours and then stimulated with 5 µg/ml of ESAT-6 protein for 24 hours. The cells were stained and subjected to flow cytometry to determine apoptosis as mentioned above in figure 4B. **P<0.01 and ***P<0.001 as compared with control vector (72 hours) +ESAT-6 group. Lower panel shows the western blot of recombinant BAT3 expressed in the cytoplasmic extracts of BAT3 plasmid transfected cells obtained at different time intervals. Total 25 µg of each protein sample was loaded in 12% SDS-PAGE gel for the development of western blot.

Ajay Grover, et al. PLoS One. 2012;7(7):e40836.
5.
Figure 1

Figure 1. Expression of BAT3 in J774A.1 cells and bone marrow-derived macrophages (BMDM) in response to non-lethal heat shock.. From: BAT3 Regulates Mycobacterium tuberculosis Protein ESAT-6-Mediated Apoptosis of Macrophages.

A. J774A.1 cells and BMDM were subjected to non-lethal heat shock at 42°C for 10 minutes and rested for 1 hour. Total protein concentration of different fractions was determined and equal amounts of proteins (25 µg) were loaded on SDS-PAGE gels. Following electrophoresis and western blotting, the blots were developed using a rabbit polyclonal antibody against BAT3 protein. The molecular weight of BAT3 was ∼122 kDa. Histogram plot shows the densitometric quantification of BAT3 protein levels shown in western blots. B. Total RNA was isolated from cells; cDNA was prepared and subjected to real-time PCR for BAT3 gene amplification. ΔΔCT values were normalized to mouse GAPDH gene. C. The exosomal pellet (Ex) and soluble fractions (SF), of both J774A.1 cells and bone marrow-derived macrophages (BMDM) culture supernatants were collected. Total 25 µg of protein from each sample was run on a 12% SDS-PAGE gel followed by western blotting to detect BAT3 (upper panel) and HSP70 (lower panel). The cytoplasmic marker GAPDH and nuclear marker histone H1 served as positive controls for the cytoplasmic extracts and nuclear extracts, respectively, in western blotting experiments.

Ajay Grover, et al. PLoS One. 2012;7(7):e40836.
6.
Figure 3

Figure 3. ESAT-6 induces the expression of BAT3 in macrophages.. From: BAT3 Regulates Mycobacterium tuberculosis Protein ESAT-6-Mediated Apoptosis of Macrophages.

A. Bone marrow-derived macrophages (BMDM) were incubated with different concentrations of ESAT-6 in Opti-MEM medium for 6 hours. Cell culture supernatants were collected and concentrated. Total protein concentration of different fractions was determined and equal amounts of proteins (25 µg) were loaded on an SDS-PAGE gel. Following electrophoresis and western blotting, the blot was developed using a rabbit polyclonal antibody against BAT3 protein. Histogram plot shows the densitometric quantification of BAT3 protein levels shown in western blot. B. BMDM were incubated with 5 µg/ml of ESAT-6. Nuclear and cytoplasmic extracts were prepared and western blots were developed as mentioned above in figure 3A. Histogram plot shows the densitometric quantification of the change in BAT3 protein levels in nuclear and cytoplasmic extracts. C. Total RNA was isolated from BMDM that were stimulated with ESAT-6 (5 µg/ml) for 6 hours; cDNA was prepared and subjected to real-time PCR for BAT3 gene amplification. ΔΔCT values were normalized to mouse GAPDH gene. D. J774A.1 cells were incubated with 5 µg/ml of ESAT-6 and western blots of nuclear and cytoplasmic fractions were developed as mentioned above in figure 3A. Histogram plot shows the densitometric quantification of the change in BAT3 protein levels in nuclear and cytoplasmic extracts. E. J774A.1 cells were stimulated with ESAT-6 (5 µg/ml) for 6 hours. Total RNA was isolated; cDNA was prepared and subjected to real-time PCR for BAT3 gene amplification. ΔΔCT values were normalized to mouse GAPDH gene. F. J774A.1 cells were incubated with 5 µg/ml of ESAT-6 and culture supernatants were collected at different time points. Heat shock-treated cell supernatants were used as positive control. Western blots were developed as mentioned above in figure 3A. Histogram plot shows the densitometric quantification of BAT3 protein levels shown in western blot. G. J774A.1 cells were incubated with 5 µg/ml of ESAT-6 and cytoplasmic extracts were collected at different time points. The western blots were developed as mentioned above in figure 3A. Histogram plot shows the densitometric quantification of BAT3 protein levels shown in western blot. H. J774A.1 cells were incubated with 5 µg/ml of Ag85B for 9 hours and different cellular fractions (Nuclear extract, Cytoplasmic extract and Supernatants) were collected at different time points. The western blots were developed as mentioned above in figure 3A. The cytoplasmic marker GAPDH and nuclear marker histone H1 served as positive controls for the cytoplasmic extracts and nuclear extracts, respectively, in western blotting experiments.

Ajay Grover, et al. PLoS One. 2012;7(7):e40836.

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