Table 1.
Primers for NF-kB in ChIP assay.
Figure 1.
SDS-PAGE electrophoresis and immunoblotting of recombinant MPT64 protein.
(A) Proteins were transferred to nitrocellulose and probed with antibody specific for MPT64, and a final detection was made using DAB substrate. (B) Proteins were stained with Coomassie Briliant Blue. Lane 1: crude lysate of k802 transformed by pGEX5T after induction. Lane 2: the expression product of k802 transduced with pGEX5T-MPT64. Lane 3: purified MPT64 protein. Lane 4: crude lysate of k802 transformed by pGEX5T after induction. Lanes 5/6: the expression product of k802 transduced with pGEX5T-MPT64, before or after induction. Lane 7: purified MPT64. Lane 8: protein markers.
Figure 2.
Apoptosis inhibition of macrophages by MPT64 protein.
PMA-differentiated RAW264.7 cells were put into 24-well flat bottom tissue culture plates at a density of 1×105 cells per well. Then, RAW264.7 macrophages were incubated with PBS, BCG-PPD (10 µg/ml) or a mixture of PPD-MPT64 at a different concentrations for 16 h. (A) Apoptosis was detected by measuring the membrane exposure of PS using annexin V by flow cytometry, and the results were analyzed. Fig. 2A Significant differences of the apoptosis percentages. *P<0.05, the PPD group vs the MPT64 (10 µg/ml) group; ** P<0.01, the PBS group vs the PPD group, and the PPD group vs the MPT64 (15 µg/ml or 20 µg/ml) group. The apoptosis percentage is not significantly different for the GST group or heat-treated MPT64 protein compared with the PPD group. Fig. 2A Ne, the PBS group; HAMPT64, heat treated MPT64; M10, MPT64 at 10 µg/ml of concentration; M15, MPT64 at 15 µg/ml of concentration; M20, MPT64 at 20 µg/ml of concentration. Fig. 2B A: The PBS group. B: The PPD group. C: The MPT64 (15 µg/ml) group. Data shown are representative of three independent experiments.
Figure 3.
Apoptosis-related gene expression after MPT64 treatment.
PMA-differentiated RAW264.7 macrophages were incubated with PBS, BCG-PPD (10 µg/ml), or a PPD-MPT64 (15 µg/ml) mixture for 16 h. Then, the bcl-2 mRNA (3A) and bax mRNA levels were detected by real-time PCR (3B) or Western-blot (3C) for the expression of bcl-2. For the Western-blot, reactive bands were detected by an ECL chemiluminescence system.*P<0.05, the PPD group vs the PPD-MPT64 (15 µg/ml) group. Fig. 3C A: The PBS group. B: The MPT64 (15 µg/ml) group. C: The PPD group. Data shown are representative of three independent experiments.
Figure 4.
MPT64 anti-apoptosis activity reduced by specific siRNA blockade for bcl-2.
siRNA for bcl-2 was synthesized and transfected into differentiated RAW264.7 cells. The cells were divided into four groups: a PBS group, a PPD group, a PPD-MPT64 (15 µg/ml) group, and a PPD-MPT64 (15 µg/ml)-siRNA group, After 16 h of transfection, the mRNA level of bcl-2 (4A) was detected by real-time PCR, and the apoptosis percentage (4B) was analyzed by flow cytometry. Fig. 4A, *P<0.05, the PPD-MPT64 (15 µg/ml)-siRNA group vs the PPD-MPT64 (15 µg/ml) group Fig. 4B, *P<0.05, the PPD-MPT64 (15 µg/ml)-siRNA group vs the PPD-MPT64 (15 µg/ml) group. Data shown are representative of three independent experiments.
Figure 5.
The control effect of miRNA21 on bcl-2 after MPT64 treatment RAW264.7.
Macrophages were treated as shown in Fig. 2. The miRNA21 level was analyzed by real time-PCR (5A). **P<0.01, the PPD group vs the PPD-MPT64 (15 µg/ml) group. The control effect of miRNA21 on bcl-2 was also analyzed by relative luciferase activity in RAW264.7 cells (5B). The wild-type or mutated type of bcl-2-utr was cloned into a pMIR-reportTM luciferase vector. Then, the recombinant plasmids and miR21 were transfected into RAW264.7 cells. The relative luciferase activity was detected. miR-mock and miR335 were used as control miRNAs. *P<0.01, the miR21 group vs the miR-mock group after the transfection by wild-type bcl-2-utr. Data shown are representative of three independent experiments.
Figure 6.
miR21 is directly regulated by NF-kB.
Real-time PCR results for miR-21 levels were performed with siRNA against NF-κB. After the transfection of siRNA against NF-κB, the miR-21 level was detected by real-time PCR (6A). **P<0.01, the siRNA treatment group vs the non- siRNA treatment group. Then, the apoptosis level was analyzed by flow cytometry (6D). **P<0.01, the siRNA treatment group vs the non- siRNA treatment group. Five primers were designed according to a software analysis, and ChIP assays were performed in RAW264.7 cells to explore possible binding sites. A specific band was observed for primer for site 3 (6B). No specific band was observed for the primers for sites 1, 2, 4 and 5 (6C). Negative controls were incubated without primary antibody. The experiment was repeated three times.