NF-κB activation leads to bendamustine resistance by increasing cFLIP expression, and ibrutinib can restore sensitivity of lymphoma cells to bendamustine. (A) Western blot analysis for cFLIP and β-actin (loading control) of HBL1, FL18, and FL318 in the absence of feeder cells or in the presence of L cells or CD40L⁺ L cells. (B) Changes in NF-κB activity of HBL1 after treatment with different concentrations of ibrutinib evaluated by luciferase reporter assay. RLU representing NF-κB activity are shown in the line graphs. Error bars represent the SEM of duplicate samples. Statistical significance between samples treated with 0 and 0.1 μM ibrutinib (upper stars) or those treated with 0 and 1 μM ibrutinib (lower stars) for 8 and 12 hours was evaluated by 2-way ANOVA, followed by Tukey-Kramer test for multiple comparisons. Results are shown as: ∗∗P < .01 or ∗∗∗P < .001. (C) Western blot analysis for cFLIP and β-actin (loading control) of HBL1 at different ibrutinib concentrations and durations of treatment. (D) Cell death rates of HBL1 cocultured with CD40L⁺ L cells treated with etoposide, 4-OHCP, and bendamustine at the indicated concentrations in combination with or without 0.1 μM ibrutinib. Error bars represent the SEM of duplicate samples. The results are indicated as: ∗∗∗P < .001. (E) Cell death rates of MINO cocultured with BAFF⁺ L cells treated with etoposide, 4-OHCP, and bendamustine at the indicated concentrations in combination with or without 0.1 μM ibrutinib. Error bars represent the SEM of duplicate samples. The results are indicated as: ∗P < .05 or ∗∗∗P < .001.