Figure 5. Effect of seliciclib on cell cycle regulators expression in hMMCLs.

(A) The indicated multiple myeloma cell lines in logarithmic growth phase were extracted and subjected to immunoblotting, utilizing CCND2, CCNE1 and p27 antibodies. β-actin expression serves as an internal loading control. (B) Seliciclib effect on CCNE1, phosphor-CCNE1, CCND2, CDK2 and p27 expression: the indicated hMMCLs were incubated in the presence or absence of 50 µM seliciclib for 4 (I) or 16 (II) hours. Control cells were incubated in the presence of DMSO. Cells were lyzed and extracts were subjected to immunoblotting, utilizing specific antibodies. (C) CAG and NCI H929 cells were incubated in the absence or presence of increasing concentration of seliciclib for over-night incubation or in 50 µM selicicilb for the indicated time points. Cells were lyzed and extracts were subjected to immunoblotting, utilizing CCND2 and CDK2 antibodies. Control cells were incubated in the presence of DMSO. Experiments were performed 3 times and one representative result is presented. (D) CAG cells were incubated in the absence or presence of seliciclib or MG132 (10 µM) exclusively or combined. p27 level of expression was verified by immunoblotting. β-actin was used to confirm equal protein loading.