Apoptosis inhibition of macrophages by MPT64 protein.

<p>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. <b>Fig. 2A</b> Significant differences of the apoptosis percentages. *<i>P</i><0.05, the PPD group vs the MPT64 (10 µg/ml) group; ** <i>P</i><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. <b>Fig. 2A</b> 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. <b>Fig. 2B</b> A: The PBS group. B: The PPD group. C: The MPT64 (15 µg/ml) group. Data shown are representative of three independent experiments.</p

miR21 is directly regulated by NF-kB.

<p>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). **<i>P</i><0.01, the siRNA treatment group vs the non- siRNA treatment group. Then, the apoptosis level was analyzed by flow cytometry (6D). **<i>P</i><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.</p

Apoptosis-related gene expression after MPT64 treatment.

<p>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.*<i>P</i><0.05, the PPD group vs the PPD-MPT64 (15 µg/ml) group. <b>Fig. 3C</b> A: The PBS group. B: The MPT64 (15 µg/ml) group. C: The PPD group. Data shown are representative of three independent experiments.</p

Primers for NF-kB in ChIP assay.

<p>Primers for NF-kB in ChIP assay.</p

The control effect of miRNA21 on bcl-2 after MPT64 treatment RAW264.7.

<p>Macrophages were treated as shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0100949#pone-0100949-g002" target="_blank">Fig. 2</a>. The miRNA21 level was analyzed by real time-PCR (5A). **<i>P</i><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. *<i>P</i><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.</p

Additional file 1 of SGOL2 is a novel prognostic marker and fosters disease progression via a MAD2-mediated pathway in hepatocellular carcinoma

Additional file 1. Supplementary methods

SDS-PAGE electrophoresis and immunoblotting of recombinant MPT64 protein.

<p>(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 <i>k802</i> transformed by pGEX5T after induction. Lane 2: the expression product of <i>k802</i> transduced with pGEX5T-MPT64. Lane 3: purified MPT64 protein. Lane 4: crude lysate of <i>k802</i> transformed by pGEX5T after induction. Lanes 5/6: the expression product of <i>k802</i> transduced with pGEX5T-MPT64, before or after induction. Lane 7: purified MPT64. Lane 8: protein markers.</p

Additional file 2 of SGOL2 is a novel prognostic marker and fosters disease progression via a MAD2-mediated pathway in hepatocellular carcinoma

Additional file 2: Fig. S1. High expression of SGOL2 in HCC in public database. Fig. S2. The mRNA and protein levels of SGOL1 after the knockdown of SGOL2 in HCC cell lines. Fig. S3. Subgroup expression analysis of SGOL2 in HCC. Fig. S4. Elevated expression of SGOL2 indicated a poor prognosis in HCC patients. Fig. S5. SGOL2 mutations and the associations between SGOL2 and immune cells in HCC. Fig. S6. Genes related to SGOL2 or MAD2 in HCC. Fig. S7. Hub gene analysis. Fig. S8. The predicted transcription factors binding to SGOL2 or MAD2

OPLS-DA scatter plots derived from ¹H NMR spectra of aqueous extracts of mouse brain tissues and validation plots from the HS and the F groups (R2X(cum)=0.84 and Q2(cum)=0.86) (A, A'

<div><p><b>), and the HF and the HS groups (R2X(cum)=0.64 and Q2(cum)=0.81) (B, B')</b>. </p> <p>The validation plots were obtained by using a permutation test that was randomly permuted for 500 times with the first component extracts. ▲ is for R2Y (cum), and ■ is for Q2 (cum). The vertical axis of the validation plots represented the R2 and Q2 values, and the horizontal axis (A', B') represented the correlation coefficients. </p></div

500-MHz ¹H NMR NOESY spectra (δ 0.5-4.7, 5.0-9.6) of aqueous extracts from brain tissues of mice in groups HF (A), HS (B), and F (C).

<p>The abbreviations of metabolites were shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0078281#pone-0078281-t001" target="_blank">Table 1</a>.</p