Yin et al. BMC Genomics 2010, 11:612 http://www.biomedcentral.com/1471-2164/11/612 Page 10 of 18 Table 4 Differentially expressed proteins identified by MALDI-TOF-MS/MS analysis of 2D gel protein profiles of iBMDCs treated with [BF/S+L/Ep] (75 ug/mL) in vitro for 12 h Spot Number Protein Name Relative Protein Levela SwissProt Accession Number Biological Function 54 34 82 26 23 98 1 58 79 109 55 88 6 41 38 78 56 13 91 70 45 192 191 Annexin A4 Peroxiredoxin 4 Vimentin Alpha-actin-2 Proliferin-1 APAF1-interacting protein beta-actin Alpha-tubulin 1, 2, 4, 6 Heat shock cognate 71 kDa protein Solute carrier family 35 member E1 Actin, cytoplasmic 1 (Beta-actin) YEATS domain-containing protein 4 Phenylalanyl-tRNA synthetase Protein disulfide-isomerase A3 [Precursor] Phosphoglycerate mutase 1 Heat shock protein 9, [Precursor] F-actin capping protein alpha subunit Cathepsin S Nucleoside diphosphate kinase Alpha-enolase Macrophage capping protein Voltage-gated potassium channel subfamily KQT member 2 Protein tyrosine phosphatase mitochondrial 1 1.73 1.56 1.13 1.12 1.1 1.09 1.05 1.04 1.04 1.04 1.03 0.96 0.92 0.92 0.87 0.84 0.83 0.81 0.74 0.73 0.63 0.46 0.34 P97429 O08807 P20152 P62739 P04095 Q9WVQ5 P99041 P68369... P63017 Q8CD26 P60710 Q9CR11 Q99M01 P27773 Q9DBJ1 P38647 P47754 O70370 Q9WV84 P17182 P24452 Q9Z351 Q66GT5 Signal transduction; cell communication Metabolism; Energy pathways Cell growth/maintenance Cell growth/maintenance Cell growth/maintenance apoptosis Cell growth/maintenance Cell growth/maintenance Protein metabolism Transport Cell growth/maintenance Regulation of nucleobase, nucleoside, nucleotide and nucleic acid metabolism Protein metabolism Protein metabolism Metabolism; Energy pathways Protein metabolism Cell growth/maintenance Protein metabolism Metabolism; Energy pathways Metabolism; Energy pathways Cell growth/maintenance Transport signal transduction; cell communication aRelative protein level with or without [BF/S+L/Ep] (75 μg/mL), 0.1% DMSO as vehicle control. intensities of the fluorescent test DCs in LPS-treated samples (Figure 5-g and 5h) are more aggregated and clumped together. In addition the colors are consider- ably brighter than those seen for the [BF/S+L/Ep]-trea- ted DCs. It was also observed that under in vitro test conditions, the individual DCs treated with LPS became enlarged in size, and thus could apparently absorb higher levels of FITC-labeled dextran and also already tended to aggregate together, as compared to DCs trea- ted with [BF/S+L/Ep]. These effects of LPS may also contribute to the observed results seen in trafficking experiments, where more aggregated and brighter DCs were detected in targeted tissues in LPS-treated DCs than in cells treated with [BF/S+L/Ep]. The potential effect of [BF/S+L/Ep] on key cellular physiological functions of DCs was further investigated in an in vitro study, where the expression of specific che- motaxis-related receptors for trafficking of DCs was ana- lyzed by flow cytometry. BMDCs with or without LPS stimulation were treated with [BF/S+L/Ep] for 24 h and then assayed for expression of the three cell surface markers CCR4, CCR5 and CCR7 (Figure 6), which are known to play an important role in the mobility of DCs, are expressed on DCs at distinguishable differentiation stages, and are located at specific sites in different tissues [23]. Here, CCR4+CCR5+ and CCR4+CCR7+ dendritic cells were considered as immature and mature DC popu- lations, respectively, as observed during their in vivo maturation processes. Our data show that in vitro treat- ment of DCs with [BF/S+L/Ep] significantly increases the expression level of CCR5 on immature DCs (Figure 6 but does not affect CCR5 expression on LPS-induced mature DCs. Furthermore, we have observed that our herbal extract treatment caused a suppressive effect on the “endogenous” or “non-LPS-induced” generation of mature DCs from immature DCs, as assessed by the CCR7 activity measured for DMSO-treated and [BF/S+L/ Ep]-treated DCs (Figure 6). Considering our prior results obtained both in vitro (i.e., DC maturation, Figure 1) and in vivo (i.e., DC trafficking capacity, Figure 5), the data shown in Figure 6 thus confirm our current key finding that the [BF/S+L/Ep]-enhanced DC trafficking ability