E. Z. N. A. Total RNA Kit www.selleckchem.com/products/epz-5676.html II for total RNA purification was from Omega. First strand cDNA synthesis kits were obtained from Invitrogen. Taqman Universal PCR Master Mix was Inhibitors,Modulators,Libraries purchased from TAKARA Bio Science and Technology Company. The LightCycler real time RT PCR System was purchased from Roche Applied Science. Rabbit mono clonal antibodies against human ApoM, ApoAI, B actin, and horseradish peroxidase conjugated goat polyclonal secondary antibody to rabbit IgG were obtained from Abcam. Cell cultures HepG2 cells were maintained in DMEM with 10% FBS in the presence of benzylpenicillin and streptomycin under standard culture condi tions. Cells were seeded in 25 cm2 cell culture flasks or in 6 well cell culture clusters and allowed to grow to 50 70% confluence.

Before the ex periment, cells were washed twice with phosphate buf fered saline and once with DMEM with 10% CTFBS. When inhibitors were used, they were added in fresh media 30 min prior to adding the other reagents. At the end of the incubation period, media were removed and saved for ApoM and ApoAI assays and the cells for determining ApoM and ApoAI mRNA levels. Effect Inhibitors,Modulators,Libraries of the androgen receptor antagonist flutamide on DHT mediated ApoM secretion and ApoM mRNA levels To evaluate whether the effect of DHT on ApoM mRNA levels and the secretion of ApoM from HepG2 cells was mediated via the androgen receptor, cells were incubated in the presence or absence of flutamide. The medium was changed when the cells grew to subconfluence, and flutamide was then added to the Inhibitors,Modulators,Libraries media.

After 30 min of incubation with flutamide, different concen trations of DHT were added, and the media and cells were harvested 24 h later for determining ApoM or ApoAI levels. Effect of protein kinase Inhibitors,Modulators,Libraries C or phosphatidylinositol 3 kinase on DHT mediated ApoM secretion and ApoM mRNA levels To evaluate whether the effect of DHT on ApoM secre tion from human HepG2 cells was mediated via protein kinase C, cells were incubated with agonist or an tagonist of PKC in the presence or absence of DHT. The medium was changed at Inhibitors,Modulators,Libraries subconfluence, after 30 min of incubation with an antagonist of the PKC superfamily or agonist of PKC, vary ing concentrations of DHT were added, and media and cells were harvested 24 h later for the determination of ApoM or ApoAI levels.

To evaluate whether the effect of DHT on ApoM secreted by HepG2 cells was mediated via phosphatidyli nositol 3 kinase, cells were incubated in the presence or absence of an inhibitor of PI3 K. After 30 min of incubation with wortmannin, Belnacasan (VX-765) different concentrations of DHT were added, and the media and cells were harvested 24 h later for the deter mination of ApoM. Mice C57BL6 J female mice were obtained from the Experi mental Animal Center of the Chinese Academy of Sciences and maintained in a 12 h12 h lightdark cycle with unlimited access to chow and water. Mice were ovariectomized at the age of 3 months and treated at the age of 7 months.