Blots were incubated with mouse anti GAPDH antibody as loading control. Kinase activity assay The kinase activity assay was performed as previously described. Briefly, 20 ng of recombinant human GSK3B were incubated with the substrate phospho glycogen synthase peptide 2, ATP and dif sellekchem ferent concentrations of PDA 66 and SB 216763 for 30 min at 30 C. After addition of Kinase Glo and 10 min of incubation at room temperature the luminescence signal was measured with a Glomax 96 microplate reader. Statistical analysis Results within each experiment were described using mean standard deviation. Significance between control and treated cells was calculated using Students t test. A p value 0. 05 was considered to be significant. The IC50 values of PDA 66 where determined with SPSS software via probit analysis.

Results PDA 66 inhibits proliferation and metabolic activity of ALL cells The influence of PDA 66 on proliferation and metabolic activity in ALL cell lines SEM, RS4,11, Jurkat and MOLT4 was analyzed by incubation with different con centrations of the drug ranging from 0. 1 uM to 10 uM for 48 and 72 h, respectively. After 48 h incu bation an inhibition of proliferation could be observed, which was even more distinct after 72 h. All cell lines showed a significant dose dependent inhibition of proliferation starting at a con centration of 0. 5 uM PDA 66. Likewise proliferation, metabolic activity decreased with increasing concentrations of PDA 66. After 72 h of incubation the metabolic activity was significantly dose dependent reduced in all cell lines starting at a concen tration of 0.

5 uM PDA 66. At this concen tration www.selleckchem.com/products/MG132.html the metabolic activity decreased to 35. 7 8. 3% in SEM, to 33. 3 4. 4% in RS4,11, to 66. 7 8% in Jurkat and to 35. 5 17% in MOLT4 cells compared to control cells treated with DMSO. Furthermore, in WST 1 assay the IC50 for PDA 66 in all four cell lines where determined. The IC50 values ranged from 0. 41 uM in SEM cells to 1. 28 uM in Jurkat cells after 72 h of incubation. The incubation of ALL cell lines with higher dosages of PDA 66 led to a decrease in cell numbers below the amount of seeded cells. This result indicates besides an inhibition of cell proliferation also an induction of cell death. PDA 66 influences morphology as well as cell cycle progression and induces apoptosis To evaluate possible morphological changes cells were treated with 1 uM of PDA 66 for 48 h and analyzed by light microscopy. All four cell lines showed similar changes in morphology after PDA 66 treatment com pared to DMSO treated control cells. Exemplarily, ef fects in SEM and Jurkat cells are shown in Figure 3.