Supplementary Figure 7: KLHL6 regulates TNFAIP3 levels in a roquin2-dependent manner.

a, Immunoblot analysis of whole-cell lysates from GFP-sorted U2932 KLHL6^+/+, KLHL6^−/− or KLHL6^−/− cells infected with lentiviruses encoding the indicated shRNAs carrying a GFP marker and treated with 10 μg ml⁻¹ of F(ab′)₂–IgM for 6 h. b, Immunoblot analysis of whole-cell lysates from GFP-sorted U2932 KLHL6^−/− (clone-derived) cells infected with retroviruses encoding empty vector (EV) or KLHL6(WT) carrying a GFP marker and treated with 10 μg ml⁻¹ of F(ab′)₂–IgM for 6 h. c, Same as in b except that cells were treated with 10 μg ml⁻¹ F(ab′)₂–IgM for the indicated times (min). d, GFP-sorted U2932 KLHL6^+/+, KLHL6^−/− or KLHL6^−/− cells infected with lentiviruses encoding the indicated shRNAs carrying a GFP marker were fractionated into cytoplasmic and nuclear extracts and analysed by immunoblotting for the indicated proteins. e, A heat map showing the presence of biallelic deletion (dark blue), monoallelic deletion (light blue) and monoallelic mutation (green) of TNFAIP3 in DLBCL tumours sequenced at UNMC⁶ and DCI⁷ (n = 1,175). Deleterious mutations of KLHL6 BTB-domain are shown in these same cases. Based on DCI database, exclusivity analysis of KLHL6 and TNFAIP3 mutations is provided in Supplementary Table 5 (n = 1,001). f, A heat map showing tumour gene alterations matched with gene expression profiling data available at UNMC. Single sample gene set enrichment analysis (GSEA) was utilized to infer NF-κB activity via expression of target gene sets from the molecular signatures database (NFkB_Q and NFkB_C)^26,59. The enrichment score is displayed as a row-normalized heat map. Unprocessed original scans of immunoblots for a–d are shown in Supplementary Fig. 8. Unless otherwise noted, immunoblots are representative of three independent experiments.