Yin et al. BMC Genomics 2010, 11:612 http://www.biomedcentral.com/1471-2164/11/612 Page 8 of 18 Table 3: Down-regulation of genes in iBMDCs treated with [BF/S+L/Ep] for (A) 4 h or (B) 12 h compared to DMSO alone (Continued) Oscar Kcne3 Sla Cluster 3 Setd4 Ndufb2 Acss1 Cluster 4 Sox6 Dmc1h Nfxl1 Huwe1 Cluster 5 Fbxo36 Cluster 6 Rbm14 Cluster 7 Speer4b Ccdc21 Mgl1 osteoclast associated receptor Voltage-gated potassium channel, Isk-related subfamily, gene 3 src-like adaptor Enzyme, Kinase SET domain containing 4 NADH dehydrogenase (ubiquinone) 1 beta subcomplex, 2 acyl-CoA synthetase short-chain family member 1 DNA binding protein, Transcription factor SRY-box containing gene 6 disrupted meiotic cDNA 1 homolog nuclear transcription factor, X-box binding-like 1 HECT, UBA and WWE domain containing 1 Protein metastasis F-box only protein 36 RNA binding protein, Translation factor RNA binding motif protein 14 Miscellaneous spermatogenesis associated glutamate (E)-rich protein 4b coiled-coil domain containing 21 macrophage galactose N-acetyl-galactosamine specific lectin 1 NM_175632 NM_020574 NM_009192 NM_145482 NM_026612 NM_080575 NM_011445 NM_010059 Q3ULV1 NM_021523 NM_025386 NM_019869 NM_028561 NM_144527 NM_010796 0.44 0.49 0.50 0.32 0.39 0.40 0.06 0.17 0.36 0.41 0.46 0.50 0.19 0.47 0.48 aExpression ratio is defined as relative difference in expression with (+) compared to without (-) treatment with [BF/S+L/Ep] plant extract; (−) is DMSO vehicle control. 4. Identification of differentially expressed known or novel proteins in BMDCs that respond to [BF/S+L/Ep] Using 2D gel electrophoresis, we were able to routinely obtain representative, high resolution, and highly repro- ducible 2D protein profiles of mouse DCs as putative proteomic maps (data not shown). Treatment of DCs with [BF/S+L/Ep] at 75 μg/mL resulted in significant changes in expression of some proteins in comparison to the solvent-treated mouse DC samples. Differentially- expressed proteins were then identified by MALDI- TOF-MS and in some cases subsequently analyzed with tandem MS/MS, after manual excision of these protein spots from gels. A total of 23 different known protein species were isolated and characterized by analysis with MALDI-TOF-MS peptide mass fingerprinting (PMF). Table 4 lists the changes in protein expression in DCs treated with [BF/S+L/Ep] in vitro for 12 h, as compared to solvent (vehicle) treatment (0.1% DMSO). Expression of metabolism, cytoskeleton or NF- B-signaling related proteins were most affected by [BF/S+L/Ep]. 2D gel electrophoretic analyses showed increased levels of a group of specific proteins, such as annexin A4 and peroxiredoxin (Figure 4A). In contrast, the levels of some other proteins, such as macrophage capping pro- tein, were reduced (Figure 4B). These proteins, which function in cytoskeleton organization, did not show a detectable change in mRNA levels after [BF/S+L/Ep] treatment. 5. Effect of [BF/S+L/Ep] extract on BMDC trafficking The effect of [BF/S+L/Ep] on DC mobility was evaluated by an in vivo DC trafficking assay. Primary BMDCs gen- erated by in vitro culture were treated with 0.1% DMSO, [BF/S+L/Ep] or LPS separately. Cells were then stained with fluorescein isothiocyanate isomer I (a green fluorescence dye) and administered to test mice via i.v. injection. After 24 h, mice were sacrificed and DC moti- lities in vivo were compared by scoring the presence and number of DCs with green fluorescence dye in tis- sue sections of inguinal lymph node, spleen and liver. As seen in Figure 5, DCs treated with [BF/S+L/Ep] showed a higher capacity to target lymph nodes and spleen (Figure 5-d, e) than DMSO-treated DCs (Figure 5a, b; Figure 6). In comparison, DCs stimulated by LPS showed a relatively higher capacity to target these immune tissues, particularly the spleen (Figure 5-g, h).