PIM2 knockdown: biomarkers and effects on apoptosis and cell cycle. (A) Western blot analysis of PIM2, p4E-BP1(Thr37/46), and p4E-BP1(Ser65) in RIVA and U2932 cell lines. Knockdown inhibition of PIM2 was achieved with 2 independent sets of siRNAs: siRNA1 (L-005359-00-0005) and siRNA2 (s21751). siRNAs with sequences that do not target any gene product (scramble) were used as controls. Bands were quantified using ImageJ 1.36b (National Institutes of Health), and the ratio of phosphorylated to total protein was calculated. The blot is representative of 2 independent experiments. (B) Induction of apoptosis and cell cycle were measured 24 and 48 hours after knockdown inhibition of PIM1, PIM2 (siRNA1 and siRNA2), PIM3, or the simultaneous inhibition of all 3 PIM family members using either PIM2-siRNA1 or PIM2-siRNA2 (T-siRNA1 or T-siRNA2, respectively). Scrambled siRNAs were used as controls. No effects on cell cycle were observed at any time or with any siRNA combination (see supplemental Table 2 for quantification of cell-cycle data).