Even so, little is known regarding the results of curcumin on other kinds of AML. While in the existing review, we investigated the result and mode of action of curcumin on monocytic leukemia THP 1 cells. We very first examined the impact of different concentrations of curcumin on THP one cell apoptosis. Upcoming, interference of the inhibitor of ERK and JNK and PMA treated THP one cells were used to research the probably mechanism of curcumin mediated apoptosis. Approaches Cell and reagents The THP 1 cell line, derived from human acute mono cytic leukemia, was obtained from American Type Culture Assortment. Cells had been cultured in RPMI 1640 supplemented with 10% FBS, 10 mM HEPES, 1% L glutamine, 1% non crucial amino acids. Curcu min, dimethyl sulfoxide, SP600125, U0126 and phorbol twelve myristate 13 acetate were bought from Sigma.
Antibo dies towards caspase three, cleaved caspase 8, Caspase 9, FoxO4, phospho FoxO4, FoxO3a, FoxO1, phos pho FoxO1, phospho FoxO3a, p85, phospho p85, p110a, PDK1, Phospho PDK1, more info here JunB, c Jun, phospho c Jun Ser63, AKT1, AKT2, AKT3, phospho AKT, phospho AKT, ATF2, phospho ATF2 Thr71, phospho JNK, phospho ERK, ERK, JNK, p38, phos pho p38, caspase eight and histone H3 were purchased from Cell signaling laboratory and anti bodies towards PARP one, caspase three and GAPDH had been from Epitomics Inc. b actin antibody and phospho JunB have been bought from Sigma and Santa Cruz Biotechnology, respectively. Movement cytometry THP 1 cells, which had been taken care of with curcumin, were harvested and fixed with 70% ethanol at 4 C overnight. Right after PBS washing, the cells have been incubated with RNase A for 5 min.
Just after incubation with propidium iodide, the cells underwent movement cytometry. For double staining, THP one cells have been initially taken care of with PhipPhiLux G1D2 cas pase 3 substrate at selleckchem 37 C for 45 min. Soon after washing, the cells have been stained with propidium iodide and analyzed working with movement cytometry. Protein extraction and immunoblotting THP 1 cells were lyzed with RIPA lysis buffer. Complete cell lysates have been extracted as described previously. The lysates had been separated working with polyacrylamide gel electrophoresis. After transfer, the membrane was blotted with antibody and developed with an enhanced chemiluminescent kit. Caspase exercise assay THP one cells had been handled with DMSO and curcumin inside the presence of U0126 and SP600125 for ten hours. The cells had been subsequently incu bated with Caspase Glo three seven reagent kit and caspases 3 7 exercise was detected and analyzed making use of a GloMax Multi Detection Technique according to your companies instructions. WST one assays THP one cells and PMA handled tHP one cells had been seeded in the density of 50 000 cells cm2 in 96 properly plates. The cells have been incubated with DMSO and 50 uM curcumin for 18 hr.