MALT1 inhibition blocks the activation of the NF-κB pathway. (A) H929 cells were treated with Mi-2 for 24 hours, and H929 cells transduced with MALT1-depletion or scramble vector were subjected to RNA-sequencing and GSEA. The enriched pathways are shown in bubble graph (false discovery rate [FDR] < 0.25; absolute value of normalized enrichment score [|NES|]> 1.4). (B) Enrichment plots of GSEA revealing significantly negative enrichments for the NF-κB pathway after MALT1 inhibition. (C) Scramble and MALT1-KO cells (IM9, RPMI8226, and U266) were subjected to WB analysis and probed with p-p65 and p65 antibodies, with GAPDH as a loading control. (D) IF analysis of -c-REL protein in scramble and MALT1-KO cells (RPMI8226 and U266). Nuclei were stained with DAPI (1 μg/mL). Bar = 10 μM. (E) WB analysis of p-IκB and IκB in scramble and MALT1-KO IM9 cells, with GAPDH as a loading control. (F) IM9 cells were treated with Mi-2 (1.5 μM) for different times (0, 0.5, 1, 2, 4, and 8 hours) and p-NF-κB was examined by WB assay. (G) IM9 cells were treated with indicated concentrations of Mi-2 for 24 hours in the absence or presence of PMA (10 ng/mL). The nuclear proteins c-REL and p65 were measured by WB assay. Histone H3 was used as a control. (H) IF analysis of p65 and c-REL protein in IM9 cells treated with Mi-2 or DMSO. Nuclei were stained with DAPI (1 μg/mL).