Figure 4.

Association of FoxM1 expression with cell cycle regulatory proteins. (A) SUDHL4 and OCI-LY19 cells were treated with and without 5 and 10 μM thiostrepton for 48 h. After cell lysis, equal amounts of proteins were separated by SDS-PAGE, transferred to Immobilon membrane, and immunoblotted with antibodies against SKP-2, p27Kip1, Aurora kinase B, Cyclin A, Cyclin B1, pRb and beta-actin as indicated. A representative of 3 different experiments is shown. (B) SKP-2 siRNA expression does not affect expression of FoxM1. OCI-LY19 cells were transfected with scrambled siRNA (100 nM) and SKP-2 siRNA (50 and 100 nM) with Lipofectamine as described in the Design and Methods section. After 48 h of transfection, cells were lysed and equal amounts of proteins were separated by SDS-PAGE, transferred to Immobilon membrane, and immunoblotted with antibodies against SKP-2, FoxM1 and beta-actin as indicated. (C) Thiostrepton treatment induces a G2/M cell cycle arrest at early time point in DLBCL. SUDHL4 and OCI-LY19 cells were treated with 10 μM thiostrepton for indicated time periods. Following incubation, cells were analyzed for cell cycle fractions by flow cytometry. (D) DLBCL cells were incubated with either 1 μM thiostrepton or 1nM bortezomib alone or in combination for 48 h. Cell viability was assayed using MTT as described in the Design and Methods section. The graph displays the mean ± SD (standard deviation) of 3 independent experiments with replicates of 6 wells for all the doses and vehicle control for each experiment *P<0.05, statistically significant (Student’s t-test.). (E) SUDHL4 cells were treated with either 1 μM thiostrepton or 1 nM bortezomib alone or in combination (as indicated) for 48 h. Cells were lysed and equal amounts of proteins were separated by SDS-PAGE, transferred to PVDF membrane, and immunoblotted with antibodies against caspase-9, caspase-3, PARP and beta-actin. (F) SUDHL4 and OCI-LY19 cells were trans-fected with either 100 nM scrambled siRNA or 25 nM specific siRNA targeted against FoxM1 alone or in combination with 1 nM bortezomib for 48 h. Cell viability was assayed using MTT as described in Design and Methods section. The graph displays the mean ± SD (standard deviation) of 3 independent experiments with replicates of 6 wells for all the doses and vehicle control for each experiment. (G) SUDHL4 cells were transfected with either 100 nM scrambled siRNA or 25 nM specific siRNA targeted against FoxM1 alone or in combination with 1 nM bortezomib for 48 h. Cells were lysed and equal amounts of proteins were separated by SDS-PAGE, transferred to PVDF membrane, and immunoblotted with antibodies against caspase-9, caspase-3, PARP and beta-actin.