POH improves the cytotoxic effects of bortezomib on CD45⁺19⁻ MCL-ICs. (A) The combination of bortezomib and POH inhibited NF-κB DNA-binding activities in CD45⁺CD19⁻ MCL-ICs compared with bortezomib. Stem-like cells (CD45⁺CD19⁻) from 5 different patients were treated with bortezomib (100nM), POH (1mM), or the combination of bortezomib (100nM) and POH (1mM) for 16 hours. Nuclear extracts from untreated, bortezomib-treated, POH-treated, and combination-treated CD45⁺CD19⁻ MCL-ICs were analyzed using ELISA assays to assess p50 and p65 DNA-binding activities. Changes in NF-κB DNA-binding activity levels before and after treatment were measured. The colorimetric values in untreated samples were subtracted from the values measured in treated samples. Results are shown as the means ± SD. *P < .05 by unpaired t test. BTZ indicates bortezomib. (B) The addition of POH to bortezomib significantly increased the cytotoxicity in CD45⁺CD19⁻ MCL-ICs. Cell viability of CD45⁺CD19⁻ primary MCL-ICs were measured after 16 hours of incubation with bortezomib (100nM), POH (1mM), or the combination of bortezomib (100nM) and POH (1mM) using 7-amino-actinomycin–stained flow cytometry. The columns represent the percentages of dead cells, and the results are shown as the means ± SD. P values were calculated using the ANOVA test and the unpaired t test. *P < .05. BTZ indicates bortezomib. (C) Live cell proportions were analyzed using 7-amino-actinomycin–stained flow cytometry with untreated, bortezomib-treated, POH-treated, and combination-treated CD45⁺CD19⁻ MCL-ICs. The combination treatement inhibited the survival of CD45⁺CD19⁻ MCL-ICs compared with bortezomib alone, POH alone, or no treatment. P values were calculated using the ANOVA test and the unpaired t test. *P < .05. BTZ indicates bortezomib. (D) The synergic cytotoxic effects of bortezomib and POH were determined using the CI based on the data from cell viability assays. CI plots were generated using CompuSyn software according to the Chou-Talalay method. The combination is synergic when CI < 1.0, additive when CI = 1, and antagonistic when CI > 1.0. POH synergized with bortezomib to induce cytotoxicity in CD45⁺CD19⁻ MCL-ICs.