Regulation of the 3′ UTR of NFKBIZ and NFKBID in DLBCL. (A-B) Schematic depiction of the 3′ UTR constructs of the (A) NFKBIZ and (B) NFKBID luciferase reporter. Relative luminescence was measured 48 hours after cotransfection of the reporter with a Renilla construct in BJAB CARD11 KOs expressing CARD11 WT, L251P, or L232LI. Values were normalized to mock vector-infected cells; n ≥ 4, all error bars depict the mean ± SD, ordinary 1-way ANOVA with Tukey multiple comparisons; ∗P < .05; ∗∗∗P < .001; and ∗∗∗∗P < .0001. (C-D) Luciferase reporter assay of the 3′ UTR of (C) NFKBIZ and (D) NFKBID in BJAB CARD11 KOs reconstituted with CARD11 L251P and L232LI, respectively, after ibrutinib, S-mepazine, or MLT-748 treatment for 18 hours. n ≥ 3, all error bars depict the mean ± SD, ordinary 1-way ANOVA with Dunnett multiple comparisons; ∗P < .05; ∗∗P < .01; ∗∗∗P < .001; and ∗∗∗∗P < .0001. (E-F) Luciferase reporter assay of the 3′ UTR reporter of (E) NFKBIZ and (F) NFKBID in ABC and GCB DLBCL cell lines. n = 3, all error bars depict the mean ± SD. (G-H) Luciferase reporter assay of the 3′ UTR of (G) NFKBIZ and (H) NFKBID in HBL1 and OCI-Ly3 after treatment with ibrutinib, S-mepazine, or MLT-748. n ≥ 4, all error bars depict the mean ± SD, ordinary 1-way ANOVA with Dunnett multiple comparisons; ∗P < .05; ∗∗P < .01; and ∗∗∗P < .001. (I) Schematic representation of the SL structure of the UTR of NFKBIZ, and location of patient-derived mutations in the UTR. (J) Luciferase reporter assay of WT and mutation variants SNV1, Del1, Del2, and Del3 UTR of NFKBIZ in BJAB CARD11 KO cells expressing CARD11 WT. The relative luminescence was normalized to the stability of the WT UTR of NFKBIZ. N = 3, all error bars depict the mean ± SD, ordinary 1-way ANOVA with Dunnett multiple comparisons; ∗∗P < .01 and ∗∗∗P < .001. (K) Luciferase reporter assay of the WT, Del1, and Del3 UTR reporter in BJAB CARD11 KO cells expressing CARD11 WT, L251P, or L232LI. n = 3, all error bars depict the mean ± SD, 2-way ANOVA with Dunnett multiple comparisons test; ∗P < .05. (L) Luciferase reporter assay of WT, Del1, and Del3 NFKBIZ UTR in a panel of ABC and GCB DLBCLs. n = 4, all error bars depict the mean ± SD, ordinary 1-way ANOVA with Dunnett multiple comparisons test; ∗P < .05; ∗∗P < .01; ∗∗∗P < .001; and ∗∗∗∗P < .0001.

Regulation of the 3′ UTR of NFKBIZ and NFKBID in DLBCL. (A-B) Schematic depiction of the 3′ UTR constructs of the (A) NFKBIZ and (B) NFKBID luciferase reporter. Relative luminescence was measured 48 hours after cotransfection of the reporter with a Renilla construct in BJAB CARD11 KOs expressing CARD11 WT, L251P, or L232LI. Values were normalized to mock vector-infected cells; n ≥ 4, all error bars depict the mean ± SD, ordinary 1-way ANOVA with Tukey multiple comparisons; ∗P < .05; ∗∗∗P < .001; and ∗∗∗∗P < .0001. (C-D) Luciferase reporter assay of the 3′ UTR of (C) NFKBIZ and (D) NFKBID in BJAB CARD11 KOs reconstituted with CARD11 L251P and L232LI, respectively, after ibrutinib, S-mepazine, or MLT-748 treatment for 18 hours. n ≥ 3, all error bars depict the mean ± SD, ordinary 1-way ANOVA with Dunnett multiple comparisons; ∗P < .05; ∗∗P < .01; ∗∗∗P < .001; and ∗∗∗∗P < .0001. (E-F) Luciferase reporter assay of the 3′ UTR reporter of (E) NFKBIZ and (F) NFKBID in ABC and GCB DLBCL cell lines. n = 3, all error bars depict the mean ± SD. (G-H) Luciferase reporter assay of the 3′ UTR of (G) NFKBIZ and (H) NFKBID in HBL1 and OCI-Ly3 after treatment with ibrutinib, S-mepazine, or MLT-748. n ≥ 4, all error bars depict the mean ± SD, ordinary 1-way ANOVA with Dunnett multiple comparisons; ∗P < .05; ∗∗P < .01; and ∗∗∗P < .001. (I) Schematic representation of the SL structure of the UTR of NFKBIZ, and location of patient-derived mutations in the UTR. (J) Luciferase reporter assay of WT and mutation variants SNV1, Del1, Del2, and Del3 UTR of NFKBIZ in BJAB CARD11 KO cells expressing CARD11 WT. The relative luminescence was normalized to the stability of the WT UTR of NFKBIZ. N = 3, all error bars depict the mean ± SD, ordinary 1-way ANOVA with Dunnett multiple comparisons; ∗∗P < .01 and ∗∗∗P < .001. (K) Luciferase reporter assay of the WT, Del1, and Del3 UTR reporter in BJAB CARD11 KO cells expressing CARD11 WT, L251P, or L232LI. n = 3, all error bars depict the mean ± SD, 2-way ANOVA with Dunnett multiple comparisons test; ∗P < .05. (L) Luciferase reporter assay of WT, Del1, and Del3 NFKBIZ UTR in a panel of ABC and GCB DLBCLs. n = 4, all error bars depict the mean ± SD, ordinary 1-way ANOVA with Dunnett multiple comparisons test; ∗P < .05; ∗∗P < .01; ∗∗∗P < .001; and ∗∗∗∗P < .0001.

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