American Society of Hematology

Figure 6

$Figure 6. Mechanisms whereby ONX0912/bortezomib combination enhances WM cell cytotoxicity. (A-B) BCWM.1 cells were treated with ONX0912 (10nM, 20nM) in presence or absence of bortezomib (2.5nM, 5nM) for 2 hours, and effects on chymotrypsin-like activity (CT-L) of the immunoproteasome (LMP7; A) and constitutive proteasome (β5; B) were evaluated by ELISA on protein lysates. Proteasome activity is expressed as fold of control (untreated cells). CalcuSyn software was used to determine presence or absence of synergism between ONX0912 and bortezomib in targeting the CT-L enzymatic activities. Combination indices (CIs) and fractions affected (FAs) of the combination of ONX0912 and bortezomib and isobolograms are shown below each panel. All experiments were repeated in triplicate. (C) BCWM.1 cells were cultured with ONX0912 (20nM, 50nM) for 48 hours, in the presence or absence of bortezomib (5nM, 10nM). Cytotoxicity was assessed by MTT assay. (D) Representative isobologram of ONX0912 and bortezomib, with the CalcuSyn software demonstrating synergy for the combination. Combination indices (CIs) and fractions affected (FAs) of the combinations of ONX0912 and bortezomib are shown. All experiments were repeated in triplicate. (E) BCWM.1 cells were cultured with ONX0912 (20nM, 50nM) in the presence or absence of bortezomib (10nM) for 12 hours. Whole-cell lysates were subjected to Western blotting using anti-PARP, –caspase-9, –caspase-3, –caspase-8, and –β-actin antibodies. (F) BCWM.1 cells were cultured with either ONX0912 (20nM), bortezomib (10nM), or the combination for 4 hours, and then TNF-α (10 ng/mL) was added for the last 20 minutes. NF-κBp65 transcription factor binding to its consensus sequence on the plate-bound oligonucleotide was studied from nuclear extracts. Wild-type and mutant are wild-type and mutated consensus competitor oligonucleotides, respectively. All results represent means ± SD of triplicate experiments. (G) BCWM.1 cells were cultured with either ONX0912 (20nM), bortezomib (10nM), or the combination for 4 hours, and TNF-α (10 ng/mL) was added for the last 20 minutes. Cytoplasmic and nuclear extracts were subjected to Western blotting using anti–p-NF-κBp65, –NF-κBp100, -nucleolin, –p-IκB, and –α-tubulin antibodies.$

Mechanisms whereby ONX0912/bortezomib combination enhances WM cell cytotoxicity. (A-B) BCWM.1 cells were treated with ONX0912 (10nM, 20nM) in presence or absence of bortezomib (2.5nM, 5nM) for 2 hours, and effects on chymotrypsin-like activity (CT-L) of the immunoproteasome (LMP7; A) and constitutive proteasome (β5; B) were evaluated by ELISA on protein lysates. Proteasome activity is expressed as fold of control (untreated cells). CalcuSyn software was used to determine presence or absence of synergism between ONX0912 and bortezomib in targeting the CT-L enzymatic activities. Combination indices (CIs) and fractions affected (FAs) of the combination of ONX0912 and bortezomib and isobolograms are shown below each panel. All experiments were repeated in triplicate. (C) BCWM.1 cells were cultured with ONX0912 (20nM, 50nM) for 48 hours, in the presence or absence of bortezomib (5nM, 10nM). Cytotoxicity was assessed by MTT assay. (D) Representative isobologram of ONX0912 and bortezomib, with the CalcuSyn software demonstrating synergy for the combination. Combination indices (CIs) and fractions affected (FAs) of the combinations of ONX0912 and bortezomib are shown. All experiments were repeated in triplicate. (E) BCWM.1 cells were cultured with ONX0912 (20nM, 50nM) in the presence or absence of bortezomib (10nM) for 12 hours. Whole-cell lysates were subjected to Western blotting using anti-PARP, –caspase-9, –caspase-3, –caspase-8, and –β-actin antibodies. (F) BCWM.1 cells were cultured with either ONX0912 (20nM), bortezomib (10nM), or the combination for 4 hours, and then TNF-α (10 ng/mL) was added for the last 20 minutes. NF-κBp65 transcription factor binding to its consensus sequence on the plate-bound oligonucleotide was studied from nuclear extracts. Wild-type and mutant are wild-type and mutated consensus competitor oligonucleotides, respectively. All results represent means ± SD of triplicate experiments. (G) BCWM.1 cells were cultured with either ONX0912 (20nM), bortezomib (10nM), or the combination for 4 hours, and TNF-α (10 ng/mL) was added for the last 20 minutes. Cytoplasmic and nuclear extracts were subjected to Western blotting using anti–p-NF-κBp65, –NF-κBp100, -nucleolin, –p-IκB, and –α-tubulin antibodies.

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