Figure 3.
Figure 3. NF-κB activation modulates ROR1-targeted treatments in MCL cell lines and primary samples. (A) Percentage of viable cells (compared with control) after 48-hour incubation with different concentrations of ROR1 2A2 mAb as indicated. Error bars represent mean plus or minus SD of triplicates. (B) Percentage of AnnexinV/PI+ cells (early and late apoptosis) and negative (live cells) after 48-hour incubation with 10 µg/mL ROR1 2A2. Data are representative of 3 independent experiments. (C) Immunoblot analysis of cytoplasmic and nuclear cell lysates of MCL cell lines plus or minus 5 µg/mL ROR1 2A2 mAb for 24 hours. (D) Percentage of viable Mino cells (compared with untreated control) pretreated or not with recombinant TNF-α (100 ng/mL) or BAFF (500 ng/mL) followed by ROR1 2A2 mAb (5 µg/mL) for 48 hours. Error bars represent mean plus or minus SD of triplicates. (E) Immunoblot analysis of NF-κB p65 and p52 levels in nuclear cell lysates of Mino cells treated as in panel D. (F) Percentage of viable cells (compared with untreated control) pretreated or not with recombinant Wnt5a (200 ng/mL) followed by ROR1 2A2 mAb (5 µg/mL) for 48 hours. Error bars represent mean plus or minus SD of triplicates. (G) Immunoblot analysis of NF-κB p65 levels in nuclear cell lysates of MCL cells treated as in panel F. (H) Percentage of viable cells (compared with untreated control) cultured alone or in coculture with L-CD40L fibroblasts plus or minus ROR1 2A2 mAb (5 µg/mL for Jeko-1 and Mino, 7.5 µg/mL for other cell lines) for 48 hours. Error bars represent mean plus or minus SD of triplicates. (I) Percentage of viable cells (compared with untreated control) of primary cells cocultured with L-CD40L fibroblasts and the cytokines cocktail plus or minus 5 µg/mL ROR1 2A2 mAb for 24 hours. Error bars represent mean plus or minus SD of triplicates. (J) Immunoblot analysis of ROR1 and NF-κB p65 levels in primary cell lysates. Cells were treated with 5 µg/mL ROR1 2A2 mAb for 24 hours. Quantifications shown for ROR1 and NF-κB p65 protein levels were normalized to β-tubulin levels and compared with untreated control for each sample. Statistical significances: *P < .05, **P < .01, ***P < .001, calculated by a 2-tailed paired Student t test.

NF-κB activation modulates ROR1-targeted treatments in MCL cell lines and primary samples. (A) Percentage of viable cells (compared with control) after 48-hour incubation with different concentrations of ROR1 2A2 mAb as indicated. Error bars represent mean plus or minus SD of triplicates. (B) Percentage of AnnexinV/PI+ cells (early and late apoptosis) and negative (live cells) after 48-hour incubation with 10 µg/mL ROR1 2A2. Data are representative of 3 independent experiments. (C) Immunoblot analysis of cytoplasmic and nuclear cell lysates of MCL cell lines plus or minus 5 µg/mL ROR1 2A2 mAb for 24 hours. (D) Percentage of viable Mino cells (compared with untreated control) pretreated or not with recombinant TNF-α (100 ng/mL) or BAFF (500 ng/mL) followed by ROR1 2A2 mAb (5 µg/mL) for 48 hours. Error bars represent mean plus or minus SD of triplicates. (E) Immunoblot analysis of NF-κB p65 and p52 levels in nuclear cell lysates of Mino cells treated as in panel D. (F) Percentage of viable cells (compared with untreated control) pretreated or not with recombinant Wnt5a (200 ng/mL) followed by ROR1 2A2 mAb (5 µg/mL) for 48 hours. Error bars represent mean plus or minus SD of triplicates. (G) Immunoblot analysis of NF-κB p65 levels in nuclear cell lysates of MCL cells treated as in panel F. (H) Percentage of viable cells (compared with untreated control) cultured alone or in coculture with L-CD40L fibroblasts plus or minus ROR1 2A2 mAb (5 µg/mL for Jeko-1 and Mino, 7.5 µg/mL for other cell lines) for 48 hours. Error bars represent mean plus or minus SD of triplicates. (I) Percentage of viable cells (compared with untreated control) of primary cells cocultured with L-CD40L fibroblasts and the cytokines cocktail plus or minus 5 µg/mL ROR1 2A2 mAb for 24 hours. Error bars represent mean plus or minus SD of triplicates. (J) Immunoblot analysis of ROR1 and NF-κB p65 levels in primary cell lysates. Cells were treated with 5 µg/mL ROR1 2A2 mAb for 24 hours. Quantifications shown for ROR1 and NF-κB p65 protein levels were normalized to β-tubulin levels and compared with untreated control for each sample. Statistical significances: *P < .05, **P < .01, ***P < .001, calculated by a 2-tailed paired Student t test.

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