Depudecin

Cell culture media and supplements such as RPMI 1640, DMEM, PBS, and antibiotics (penicillin, streptomycin, and puromycin) were purchased from Lonza (Allendale, NJ), Fisher Scientific (Pittsburgh, PA) or Santa Cruz Biotechnology (Dallas, TX). Heat-inactivated fetal bovine serum (FBS) was obtained from Atlanta Biologicals (Flowery Branch, GA). Sodium butyrate and sanguinarine were procured from Sigma-Aldrich (St. Louis, MO). Trichostatin A (TSA), apicidin, HC toxin, LY294002, PX866, CAL-101, MK2206, Triciribine, GDC0068, Rapamycin, AZD8055, and BEZ235 were obtained from Cayman Chemical (Ann Arbor, MI). Tetrandrine was acquired from Santa Cruz Biotechnology, and (–)-depudecin was purchased from BioVision (Milpitas, CA) and MyBioSource (San Diego, CA). Datiscetin was ordered from BOC Sciences (Shirley, NY), while wortmannin and CUDC-907 were procured from Selleck Chemicals (Houston, TX).

The compounds with a normalized SSMD value of no less than 3.0 were further assayed for their luciferase activity in the stable IPEC-J2/PBD3-luc cell line at three different concentrations in 96-well plates. After normalization to the cell viability, the fold change in the luciferase activity of each compound relative to the nonstimulation control was calculated. Compounds showing a minimum 5-fold increase at any of the three concentrations examined were further evaluated in the parental IPEC-J2 cell line and a porcine lung alveolar macrophage cell line 3D4/31 for their ability to induce the mRNA expression of porcine HDP genes. All individual compounds were purchased from Cayman Chemical (Ann Arbor, MI), except for (−)-depudecin, which was procured from BioVision (Milpitas, CA) and MyBioSource (San Diego, CA). Three different concentrations of each compound were applied to the cells in 12-well plates for 24 h, followed by total RNA isolation, reverse transcription, and real-time PCR as described below. All treatments were performed in duplicate and repeated 2–4 times.