ght for possible development of these phytocompounds as defined therapeutic agents. We also Dorsomorphin suggest that specific and structurally different phyto compounds extracts may exert their immune modula tory effects through recruitment of a number of common signaling networks of immune responsive genes that warrant future systematic investigation. Methods Cell culture and monocyte preparation The human myelogenic leukemia cell line THP 1 was purchased from American Type Culture Collection. Cell cultures were maintained in RPMI 1640 supplemented with 10% fetal bovine serum, penicillin, and strepto mycin at 37 C in 5% CO2 in a humidified incubator. Preparation of phytocompounds Shikonin was purchased from TCI, emo din was purchased from Acros Organics, and cytopiloyne was isolated as described previously and provided by the Metabolomics Core Laboratory of the Agricultural Biotechnology Research Center, Aca demic Sinica.

Cell viability assay Cell viability was assayed by MTT colorimetric dye reduction method as described previously. Extracts or phytocompounds tested were serially diluted, shiko nin, emodin, cytopiloyne, BF S L Ep. Throughout our experiments, LPS was used at 1 ug ml in test culture medium for sti mulation of THP 1 cells. RNA isolation 1 �� 107 THP 1 cells were transferred to a 10 cm Petri dish in 10 ml culture medium. After incubation over night, test phytocompounds and LPS were added, and cells were then harvested at different time points. THP 1 cells were collected and pelleted in a microcentrifuge at 900 rpm, and the culture medium supernatant removed.

Pelleted THP 1 cells were lyzed with Trizol reagent and extracted with chloroform. The upper aqueous phase was collected by centrifugation at 4 C, 14000 rpm for 15 min utes, and RNA was precipitated from solution by the addition of an equal volume of isopropanol. RNA pellets were washed twice with 75% ethanol DEPC, and dis solved in DEPC treated water. Concentration and quality of the RNA samples was analyzed by absorbance at 260 280 nm, before they were stored at 80 C. RNA electrophoresis Aliquots of 2 ul RNA sample were added to 10 ul of a glyoxal reaction mixture in Cilengitide a closed microcentrifuge tube, incubated at 55 C for 1 hour and then chilled on ice for 2 min, when the aqueous dro plets condensed on the wall of the microcentrifuge tube were spun down.

RNA samples made up in 1�� BTPE buffer were loaded onto a 6 cm long 1% agarose gel, and electrophoresed in 1�� BTPE buffer at 100 V for 15 minutes. Gels were photographed www.selleckchem.com/products/Vorinostat-saha.html without additional staining. RT PCR reactions used the AccessQuick RT PCR system according to the manufacturers instruc tions. Briefly, 1 ug of total RNA from each sample was added to the reaction mixture containing 1�� Access Quick master mix, 10 uM each of specific sense and anti sense primers, 5U AMV reverse transcriptase, and nuclease free water to a final volume of 50 ul. Reactions were incubated at 48 C for 60 min, and PCR amplification was carried out after denaturing a