American Society of Hematology

Figure 4.

$DMF potently inhibits IKK activity. (A) Heatmaps of differentially expressed genes in HBL-1 cells treated with DMF for 12, 24, 36, and 48 hours compared with the solvent control. Gene expression changes are depicted according to the color scale. (B) Gene set enrichment analysis identified a gene set describing NF-κB targets to be downregulated by DMF treatment. (C) The indicated ABC DLBCL cell lines were treated with solvent or 20 μM DMF for 6 hours. Transcript levels of TNFAIP3, CD40, BIRC3, and NFKBIA were quantified by quantitative polymerase chain reaction. Expression of DMF-treated cells was normalized to the respective solvent control. SDHA served as reference gene. (D) ABC DLBCL cell lines were treated with 40 μM DMF for 4 hours. S32/36 phosphorylation of IκBα was visualized by immunoblot analysis. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) served as loading control. (E) After treatment with either solvent or DMF for 16 hours, nuclear fractions were isolated and binding of the NF-κB members RelA, c-Rel, and p50 to their consensus nucleotide sequence was quantified by TransAM assay. (F) HBL-1 cells were treated with the indicated DMF concentrations or with the MALT1 inhibitor LVSR-fmk (2 μM) for 4 hours in combination with the proteasome inhibitor MG-132 (5 μM) 90 minutes before cell lysis. MALT1-mediated RelB cleavage was visualized by immunoblot analysis. GAPDH served as loading control. (G) HBL-1 cells were treated with solvent, 40 μM DMF, or 40 μM DMS for 4 hours. After lysis, proteins containing reactive cysteine residues were labeled with biotin-coupled iodoacetamide (IA-biotin) and subsequently pulled down with streptavidin (SA) agarose. The accessibility of cysteine residues in IKK2 and KEAP1 was analyzed by immunoblotting. (H) HEK293T cells were transfected with the indicated IKK2 constructs, treated for 1 hour with solvent or DMF, and analyzed for IκBα phosphorylation by immunoblotting. GAPDH served as loading control. Data are representative of ≥2 (E,G) or ≥3 (C-D,F,H) independent experiments. Statistical significance was calculated using the Student t test. *P < .05, **P < .01, ***P < .001. mRNA, messenger RNA.$

DMF potently inhibits IKK activity. (A) Heatmaps of differentially expressed genes in HBL-1 cells treated with DMF for 12, 24, 36, and 48 hours compared with the solvent control. Gene expression changes are depicted according to the color scale. (B) Gene set enrichment analysis identified a gene set describing NF-κB targets to be downregulated by DMF treatment. (C) The indicated ABC DLBCL cell lines were treated with solvent or 20 μM DMF for 6 hours. Transcript levels of TNFAIP3, CD40, BIRC3, and NFKBIA were quantified by quantitative polymerase chain reaction. Expression of DMF-treated cells was normalized to the respective solvent control. SDHA served as reference gene. (D) ABC DLBCL cell lines were treated with 40 μM DMF for 4 hours. S32/36 phosphorylation of IκBα was visualized by immunoblot analysis. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) served as loading control. (E) After treatment with either solvent or DMF for 16 hours, nuclear fractions were isolated and binding of the NF-κB members RelA, c-Rel, and p50 to their consensus nucleotide sequence was quantified by TransAM assay. (F) HBL-1 cells were treated with the indicated DMF concentrations or with the MALT1 inhibitor LVSR-fmk (2 μM) for 4 hours in combination with the proteasome inhibitor MG-132 (5 μM) 90 minutes before cell lysis. MALT1-mediated RelB cleavage was visualized by immunoblot analysis. GAPDH served as loading control. (G) HBL-1 cells were treated with solvent, 40 μM DMF, or 40 μM DMS for 4 hours. After lysis, proteins containing reactive cysteine residues were labeled with biotin-coupled iodoacetamide (IA-biotin) and subsequently pulled down with streptavidin (SA) agarose. The accessibility of cysteine residues in IKK2 and KEAP1 was analyzed by immunoblotting. (H) HEK293T cells were transfected with the indicated IKK2 constructs, treated for 1 hour with solvent or DMF, and analyzed for IκBα phosphorylation by immunoblotting. GAPDH served as loading control. Data are representative of ≥2 (E,G) or ≥3 (C-D,F,H) independent experiments. Statistical significance was calculated using the Student t test. *P < .05, **P < .01, ***P < .001. mRNA, messenger RNA.

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