The next day, the cells were selleck chemicals washed with PBS, stained with 50 mg/mL propidium iodide, and dissolved in 100 mg/L RNase A. The sub G1 peak and cell cycle distribution were measured with Cytomic FC 500 and analyzed using Modifit LT software. Western blot analysis Exponentially growing K562 cells treated with various concentrations of lycorine or with out lycorine were cultivated at 5 105 cells/mL in sev eral culture flasks. After 24 h of culture, the cells were pelleted by centrifugation, washed three times with PBS, resuspended in 100 uL of RIPA lysis buffer, and centrifuged at 13000 rpm and 4 C for 15 min to collect the supernatant. The supernatant protein concentration was measured using a bicinchoninic acid protein assay kit.

Equal amounts of protein from each group were electrophoresed for 2 h on 10% sodium dodecyl sulfate polyacrylamide gels and then transferred to a PVDF membrane using an electroblotter for 100 min at 4 C. Membranes were blocked in PBS with 0. 1% Tween 20 containing 5% non fat dried milk power for 1 h. An antibody raised against tubulin, an antibody raised against pRB, an antibody raised against p21 an antibody raised against phos phorylated pRB, and antibodies raised against p53, cyclin D1, CDK4, and CDK2 were diluted in PBST containing 5% non fat milk and membranes were incu bated overnight at 4 C. After washing four times with PBST for 10 min each time, the blot was incubated with anti mouse or anti rabbit IgG conjugated with horserad ish peroxidase for 1 h at room temperature.

After washing three times with PBST for 10 min each time, the blots were developed with a chemiluninescene detection kit, and the optical density of each band was quantified by densitometric scanning. Statistical analysis The statistical difference between groups was deter mined by AVOVA and Tukeys studentized range test. Differences among groups were considered statistically different at P 0. 05. Background The serine/threonine kinase, Mirk/Dyrk1B is expressed in few normal tissues, but in skeletal muscle and many types of human cancers. Mirk/Dyrk1B has the ability to auto phosphorylate on tyrosine activating itself and then phosphorylate other substrates on serine and threo nine. therefore, it has been categorized as a dual func tion kinase.

One role of Mirk/Dyrk1B in skeletal muscle differentiation after a stress signal of serum deprivation is to block cycling myoblasts in the G0 quiescent state by phosphorylation of the cell cycle regulators cyclin D1 and CDK inhibitor p27kip1. Specificially, phos phorylation by Mirk/Dyrk1B at a conserved Entinostat ubiquitina tion site Thr288 initiates proteolysis of cyclin D1, while p27kip1 was stabilized following phosphorylation by Mirk/Dyrk1B at Ser10. As normal cells in quiescence ac tivate pathways that protect them from metabolic stress, the subpopulation of tumor cells is likely to utilize similar pathways to survive within the tumor micro environment.