S14161 induced cell death and apoptosis in myeloma and leukemia cells and primary patient samples. (A) Acute myeloid leukemia (AML) patient samples (n = 5) and normal hematopoietic cells (peripheral blood stem cells [PBSC]) were treated with increasing concentrations of S14161. Seventy-two hours after incubation, cell growth and viability were measured by the MTS assay. Data represent the mean percentage of viable cells ± SD from experiments performed in triplicate. (B) Leukemia (HL60 and U937) and myeloma (JJN3 and KMS11) cells were treated with S1 (5μM) or vehicle control. Twenty-four hours after treatment, apoptosis was measured by annexin V staining. A representative experiment is shown. FITC indicates fluorescein isothiocyanate. (C) Myeloma cells OCI-My5 (left) were incubated with 5μM S1 for the indicated time. After incubation, cells were harvested and total proteins isolated. Cleavage of poly(ADP ribose) polymerase (PARP) and caspase-9 (Casp-9) was measured by immunoblotting. Right, OCI-AML2 cells were treated for 24 hours at indicated concentrations, followed by evaluation of PARP and caspase-3 (Casp-3). (D) Myeloma (OPM2) cells were treated with increasing concentrations of S14161 (S1) for 24 hours. After incubation, cells were harvested and total proteins isolated. Expression of Bim, Bcl-2, Mcl-1, and loading controls β-actin and GAPDH was measured by immunoblotting. (E) Leukemia cells K562 were transfected with cyclin D2 with nanoparticles used as vectors (IR). Twenty-four hours later, cells were harvested for cyclin D2 (CCND2) evaluation (right) or further treated with S14161 (S1) for 24 hours followed by caspase-3 (Casp-3) activation analysis with caspase-3 specific antibody. Both pro-Casp-3 and cleaved Casp-3 fragments were detected. GAPDH was used as a loading control.