data for fig and table

data for fig and tabl

Proteomic Investigation into Betulinic Acid-Induced Apoptosis of Human Cervical Cancer HeLa Cells

<div><p>Betulinic acid is a pentacyclic triterpenoid that exhibits anticancer functions in human cancer cells. This study provides evidence that betulinic acid is highly effective against the human cervical cancer cell line HeLa by inducing dose- and time-dependent apoptosis. The apoptotic process was further investigated using a proteomics approach to reveal protein expression changes in HeLa cells following betulinic acid treatment. Proteomic analysis revealed that there were six up- and thirty down-regulated proteins in betulinic acid-induced HeLa cells, and these proteins were then subjected to functional pathway analysis using multiple analysis software. UDP-glucose 6-dehydrogenase, 6-phosphogluconate dehydrogenase decarboxylating, chain A Horf6-a novel human peroxidase enzyme that involved in redox process, was found to be down-regulated during the apoptosis process of the oxidative stress response pathway. Consistent with our results at the protein level, an increase in intracellular reactive oxygen species was observed in betulinic acid-treated cells. The proteins glucose-regulated protein and cargo-selection protein TIP47, which are involved in the endoplasmic reticulum pathway, were up-regulated by betulinic acid treatment. Meanwhile, 14-3-3 family proteins, including 14-3-3β and 14-3-3ε, were down-regulated in response to betulinic acid treatment, which is consistent with the decrease in expression of the target genes <i>14-3-3β</i> and <i>14-3-3ε</i>. Furthermore, it was found that the antiapoptotic <i>bcl-2</i> gene was down-regulated while the proapoptotic <i>bax</i> gene was up-regulated after betulinic acid treatment in HeLa cells. These results suggest that betulinic acid induces apoptosis of HeLa cells by triggering both the endoplasmic reticulum pathway and the ROS-mediated mitochondrial pathway.</p></div

Table_2_Metabolomics of Early Stage Plant Cell–Microbe Interaction Using Stable Isotope Labeling.pdf

<p>Metabolomics has been used in unraveling metabolites that play essential roles in plant–microbe (including pathogen) interactions. However, the problem of profiling a plant metabolome with potential contaminating metabolites from the coexisting microbes has been largely ignored. To address this problem, we implemented an effective stable isotope labeling approach, where the metabolome of a plant bacterial pathogen Pseudomonas syringae pv. tomato (Pst) DC3000 was labeled with heavy isotopes. The labeled bacterial cells were incubated with Arabidopsis thaliana epidermal peels (EPs) with guard cells, and excessive bacterial cells were subsequently removed from the plant tissues by washing. The plant metabolites were characterized by liquid chromatography mass spectrometry using multiple reactions monitoring, which can differentiate plant and bacterial metabolites. Targeted metabolomic analysis suggested that Pst DC3000 infection may modulate stomatal movement by reprograming plant signaling and primary metabolic pathways. This proof-of-concept study demonstrates the utility of this strategy in differentiation of the plant and microbe metabolomes, and it has broad applications in studying metabolic interactions between microbes and other organisms.</p

Flow cytometric analysis of reactive oxygen species (ROS) in BA-treated cells.

<p>HeLa cells were treated with 15 µmol/L, 30 µmol/L BA for 48 h and then incubated with 10 mmol/L DCFH-DA for 40 min. The fluorescent intensity of DCFH was measured by flow cytometry. (A) Actual spectra from a representative single experiment. (B) The fluorescence intensity of stained cells was determined by flow cytometry. Columns show mean values of three experiments (±SD). **<i>p</i><0.01 compared with the control group (0 µmol/L BA).</p

The mRNA expression of <i>14-3-3β</i>, <i>14-3-3ε</i>, <i>Bcl-2</i>, <i>Bax</i> were detected by qRT-PCR.

<p>HeLa cells were treated with 30 µmol/L BA for 24 h and then Total RNA was isolated using a Trizol reagent. Columns show mean values of three experiments (±SD). *<i>p</i><0.05, **<i>p</i><0.01 compared with the control group (0 µmol/L).</p

Effects of BA on the proliferation and apoptosis of HeLa cells.

<p>(A) Effect of BA towards Hela cells as determined by the MTT assay. Cells were treated with concentrations of BA (0 µmol/L, 15 µmol/L, 30 µmol/L, 50 µmol/L) for indicated time (24 h, 48 h, 72 h). The values for each BA concentration tested represent mean of three experiments, datas are presented as mean average ± SD; **<i>p</i><0.01 compared with the control group (0 µmol/L BA). (B) Hoechst 33258-staining of HeLa cells treated with 15 µmol/L, 30 µmol/L BA. Red arrows indicate several apoptotic cells with typical condensation of chromatin.</p

Oligonucleotides and expected sizes of PCR products for qRT-PCR analysis of differentiation marker genes.

<p>Oligonucleotides and expected sizes of PCR products for qRT-PCR analysis of differentiation marker genes.</p

Proteins identified for which the level of expression changed significantly (<i>p</i><0.05) after the treatment of 30 µmol/L BA for 48 h.

a<p>Assigned spot number as indicated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0105768#pone-0105768-g002" target="_blank">Fig. 2</a>.</p>b<p>Each protein spot was identified based on mass spectra of tryptic peptides obtained by MALDI-TOF mass spectrometry.</p>c<p>Abbreviate name is connected with signal network.</p>d<p>Accession numbers of the identified protein spots were obtained from the NCBInr-human data base.</p>e<p>Theoretical molecular mass (Da) and pI.</p>f<p>Mascot score reported after searching against the NCBInr database.</p>g<p>The protein spots altered over 1.5-folds.</p

Image_3_Metabolomics of Early Stage Plant Cell–Microbe Interaction Using Stable Isotope Labeling.TIF

<p>Metabolomics has been used in unraveling metabolites that play essential roles in plant–microbe (including pathogen) interactions. However, the problem of profiling a plant metabolome with potential contaminating metabolites from the coexisting microbes has been largely ignored. To address this problem, we implemented an effective stable isotope labeling approach, where the metabolome of a plant bacterial pathogen Pseudomonas syringae pv. tomato (Pst) DC3000 was labeled with heavy isotopes. The labeled bacterial cells were incubated with Arabidopsis thaliana epidermal peels (EPs) with guard cells, and excessive bacterial cells were subsequently removed from the plant tissues by washing. The plant metabolites were characterized by liquid chromatography mass spectrometry using multiple reactions monitoring, which can differentiate plant and bacterial metabolites. Targeted metabolomic analysis suggested that Pst DC3000 infection may modulate stomatal movement by reprograming plant signaling and primary metabolic pathways. This proof-of-concept study demonstrates the utility of this strategy in differentiation of the plant and microbe metabolomes, and it has broad applications in studying metabolic interactions between microbes and other organisms.</p

Data_Sheet_1_Metabolomics of Early Stage Plant Cell–Microbe Interaction Using Stable Isotope Labeling.xlsx

<p>Metabolomics has been used in unraveling metabolites that play essential roles in plant–microbe (including pathogen) interactions. However, the problem of profiling a plant metabolome with potential contaminating metabolites from the coexisting microbes has been largely ignored. To address this problem, we implemented an effective stable isotope labeling approach, where the metabolome of a plant bacterial pathogen Pseudomonas syringae pv. tomato (Pst) DC3000 was labeled with heavy isotopes. The labeled bacterial cells were incubated with Arabidopsis thaliana epidermal peels (EPs) with guard cells, and excessive bacterial cells were subsequently removed from the plant tissues by washing. The plant metabolites were characterized by liquid chromatography mass spectrometry using multiple reactions monitoring, which can differentiate plant and bacterial metabolites. Targeted metabolomic analysis suggested that Pst DC3000 infection may modulate stomatal movement by reprograming plant signaling and primary metabolic pathways. This proof-of-concept study demonstrates the utility of this strategy in differentiation of the plant and microbe metabolomes, and it has broad applications in studying metabolic interactions between microbes and other organisms.</p