NSC697923 inhibits constitutive NF-κB signaling, proliferation, and survival in ABC-DLBCL cells. (A) Inhibition of the Ubc13∼Ub conjugate formation in DLBCL cells by NSC697923. OCI-Ly10 cells were treated with the indicated concentrations of NSC697923 for 3 hours. The levels of Ubc13∼Ub conjugate were analyzed by Western blotting using a Ubc13-specific Ab as described in Figure 4E. In the figure, the leftmost lane was moved and placed next to the rest of the lanes on the same gel for easy comparison. (B) NSC697923 inhibits constitutive NF-κB activation in ABC-DLBCL cells. OCI-Ly10 cells were treated with the indicated concentrations of NSC697923 for 3.5 hours. The levels of the indicated proteins were analyzed by Western blotting with the respective Abs. (C) NSC697923 inhibits the proliferation and survival of ABC-DLBCL cells. The indicated ABC-DLBCL cells were seeded at 3 × 10⁵ cells/mL in 6-well plates and cultured in the presence of DMSO (0.2%, control) or various concentrations of NSC697923 for 24 hours. The live and dead cells were counted using the trypan blue exclusion assay. Shown are the means from 3 separate experiments. The average number of live cells in the control was set at 100. (D) OCI-Ly10 cells were treated with the indicated concentrations of NSC697923 for 5 hours. The levels of the indicated proteins were analyzed by Western blotting. For the detection of caspase-3, a mixture of a caspase-3 Ab and an Ab specific for the cleaved form of this protein was used. (E) OCI-Ly10 cells were treated as in panel C. The apoptotic cells (annexin V–positive cells) were measured by flow cytometry as described in “Analyses of cell viability, apoptosis, and cell-cycle progression.” A representative result from 3 independent experiments is depicted. (F) OCI-Ly10 cells were treated with the indicated concentrations of NSC697923 for 4.5 hours. Bromodeoxyuridine was then added into the culture medium for 30 minutes before the cells were harvested for cell-cycle distribution analysis as described in “Analyses of cell viability, apoptosis, and cell-cycle progression.” Shown are the averages from 2 independent experiments.