Silencing of CKS1B induces apoptosis independent of p27^Kip1 regulation in KMS28PE cell line. (A) CKS1B, SKP2, and CDKN1B (p27^Kip1) mRNA expression was evaluated by microarray analysis 5 days after lentiviral infection in KMS28PE cells expressing a nonspecific scrambled shRNA (SCRAM), CKS1B shRNA, or SKP2 shRNA. Note that CDKN1B expression is absent in KMS28PE, compared to the mean expression value of the other cell lines shown in Figure 2A-C. All experiments were performed in duplicate and the results were expressed as the mean ± standard error. (B) Western blot analysis on nuclear fractions of the same aliquots of cells shown in panel A confirmed that CKS1B shRNA and SKP2 shRNA efficiently inhibited CKS1B and SKP2 protein expression, respectively, and did not increase p27^Kip1 levels that remained absent. Cullin 1A (Cul1A) expression was used as control for the specificity of CKS1B and SKP2 action. p27^T187A overexpression was confirmed in KMS28PE 5 days after lentiviral infection, compared to the control cells infected with an empty vector (EV). Histone 1 was used as a loading control. Effects of CKS1B or SKP2 shRNA and p27^T187A overexpression on (C) cell proliferation and (D) cell viability in KMS28PE. (E) Cell cycle distribution and apoptosis were evaluated by flow cytometry analysis performed 5 days after lentiviral infection in KMS28PE cells expressing a scrambled sequence (SCRAM), CKS1B shRNA, SKP2 shRNA, or p27^T187A cDNA. Note that silencing of CKS1B induced a dramatic increase in the percentage of cells with sub-G₀ DNA content (indicative of apoptosis); SKP2 shRNA caused a modest increase of apoptotic cells, and overexpression of p27^T187A had no affect on apoptosis but resulted in a significantly higher percentage of cells in G₂-M phase. (F) Caspase-3 (active form, p17) and PARP activation was evaluated by Western blot analysis performed in the same aliquots of cells used in panel E. The following conditions were analyzed: (1) positive control (myeloma cells treated with bortezomib at 10 nM for 24 hours); (2) positive control + Z-VAD-fmk; (3) negative control (myeloma cells without any treatment); (4) CKS1B shRNA; (5) CKS1B shRNA + Z-VAD-fmk; (6) SCRAM; (7) SCRAM + Z-VAD-fmk; (8) empty vector; (9) p27^T187A cDNA; (10) SKP2 shRNA. Note that the activation of caspase-3 and PARP in cells expressing CKS1B shRNA could be abrogated by the pretreatment with the pan-caspase inhibitor Z-VAD-fmk, indicating a caspase-dependent mechanism of apoptosis.