Yin et al. BMC Genomics 2010, 11:612 http://www.biomedcentral.com/1471-2164/11/612 phosphoglycerate mutase 1, cathepsin S, nucleoside dipho- sphate kinase and alpha-enolase were also affecteded by [BF/S+L/Ep] treatment (Table 4). Whether these changes are functionally related to specific cellular and molecular activities of mouse DCs needs to be further investigated. Previous studies have demonstrated that specific alky- lamides of E. purpurea can effectively stimulate macro- phage function in healthy rats or RAW 264.7 cells [40,41]. In addition, specific in vitro effects of Echinacea extract and several of its phytochemical components on NF- B expression in Jurkat cells (a human T-cell line) have been reported [42]. Recently we reported that phy- tocompounds from E. purpurea extracts could affect specific cell-surface marker expressions and cytoskeleton rearrangement in treated human DCs [9,10]. These find- ings suggest that a spectrum of specific immunomodula- tory effects in DCs can be modulated by phytochemicals from E. purpurea at the cellular level. It is interesting to find that in this study, although the specific groups of responsive RNA transcripts and proteins seem to differ substantially from the results obtained from our pre- vious studies on human DCs, the end result in terms of cellular functions seems to suggest that many of them, such as cytoskeleton-associated gene or protein expres- sion can in fact be quite similar, and this provides valu- able correlated information for future evaluations of the various molecular mechanisms of these effects. The in vivo DC trafficking results obtained in this study may further suggest the importance of the mobility and cytoskeletal components of the differentially expressed transcriptome for future evaluation of pharmaco-geno- mics activities in a mouse DC system in vivo. Besides, we believe that our current transient labeling system for DCs may provide future application to translational research on phytochemical or herbal medicinal effect on DCs. Conclusion In this study, data obtained from both in vitro transcrip- tomic analyses and in vivo DC trafficking assays suggested that [BF/S+L/Ep] treatment can affect DC mobility. The effects of this bioactive herbal extract on expression of cytoskeleton-related RNA transcripts as well as on differ- ential protein expressions related to cell adherence and other DC functions add complementary and supportive information to our previous studies on human DCs [9,10]. Taken together, our research shows that this candidate herbal medicine may cause drastic modulatory effects on specific immune cells (DCs) by regulating key cellular behaviors. In addition, bioinformatics studies from this and our previous study [10] have revealed a group of can- didate target molecules and signaling networks, for exam- ple the JNK, PP2C-a, AKT and MAPKAPK pathways, that we believe warrant future systematic studies as key targets Page 15 of 18 for immunomodulatory phytomedicines, via mediation with dendritic cells. Methods Plant materials and plant extract preparations and fractionations Echinacea purpurea plants grown to the flowering stages were harvested from a reputable organic farm in Puli, Nantou County, Taiwan as previously described [9]. Stem and leaf tissues of fresh plants were extracted at room temperature by imbibition in 70% aqueous ethanol for 35 days, mimicking a traditional Chinese medicine (TCM) preparation protocol. The final 70% ethanol- soluble fraction was dried under vacuum, re-suspended and dissolved in 1 liter of water and then successively par- titioned with ethyl acetate (1 L × 3 times) and n-butanol (1 L × 3 times) to yield three subfractions designated as the EA, BuOH and water (H2O) fractions of the (S+L) extract [10,43]. The percentage yield for the BuOH frac- tion of the stem and leaf tissue extract [BF/S+L/Ep] was 7.23% of the 70% ethanol extracts by dry weight. Culture of DCs from bone marrow DCs were generated by using the method described by Inaba et al. [44]. Six-week-old female BALB/c (H-2Kd, I-Ad) mice were purchased from the National Labora- tory Animal Center and kept under specific pathogen free (SPF) conditions. Femurs and tibiae were removed after euthanasia and the bone marrow was flushed with RPMI-1640 medium using a syringe with a 0.45-mm needle. Red blood cells in suspension were lysed with ACK lysing buffer (150 mM NH4Cl, 1.0 mM KHCO3, 0.1 mM EDTA) for 5 min. Bone marrow cells were sus- pended at concentration of 1×107cells/30 ml complete media (CM, RPMI-1640 supplemented with 10% fetal bovine serum (FBS), 2 mM L-glutamine, 1% of nones- sential amino acids and 100 U/mL penicillin and 100 μg/mL streptomycin). Test cells were cultured for up to 9 days with 1000 U/mL of GM-CSF at 37°C, 5% CO2. On day 3, two-thirds of the medium was removed and 30 mL fresh medium with GM-CSF was added to the cells. On day 6, culture plates were gently swirled and the floating and loosely adherent cells were discarded. Aliquots of 75% of culture media were replaced with fresh culture medium with GM-CSF. On day 9, non- adherent cells were collected and used as the immature DC population for subsequent tests and analyses. Treatment of DCs with E. purpurea extract [BF/S+L/Ep] was dissolved in 100% pure and endotoxin- free dimethyl sulfoxide (DMSO). The working concen- trations of each extract sample were prepared by serial dilutions to 75 μg/mL. This dosage for stimulation of DCs was chosen based on our results obtained by MTT