RNF138 is required for NF-κB activation and tumor growth in MYD88^L265P lymphoma cells. (A-B) PLA to analyze the interaction between MYD88^L265P and IRAK-1 (A) or IRAK-4 (B) in TMD8 cells with RNF138 knockdown. ***P < .001. The bars represent 5 μm. WGA, Alexa Fluor 488 (green); PLA, Alexa Fluor 568 (red). (C) IB analysis of NF-κB and STAT3 signaling in RPCI.WM1, TMD8, and HBL-1 cells with RNF138 knockdown. (D) IB analysis of p65 in cytosolic (cyto) and nuclear fractions from RPCI.WM1 and TMD8 cells with RNF138 knockdown. (E) Real-time polymerase chain reaction analysis of NF-κB downstream target genes in RPCI.WM1, TMD8, HBL-1, and U2932 cells after RNF138 knockdown. (F-G) MTS (F) and colony-forming unit (G) assays to examine cell viability and colony formation of RPCI.WM1 cell. ***P < .001. The bar represents 50 μm. (H-J) Effect of RNF138 knockdown on tumor growth of RPCI.WM1 cells in NSG mice. Tumor volumes were monitored (H), and tumors were dissected and weighed (I). The bar represents 1 cm. (J) Ki67 was examined using IHC analysis. Ki67 was stained with DAB as the chromogen (brown). The bar represents 25 μm. ***P < .001. (K) Kaplan-Meier curve analysis of the survival of mice bearing RPCI.WM1 xenograft tumors. ***P < .001. (L) IB analysis of RNF138 protein levels in individual RPCI.WM1-derived xenograft tumors. WCE, whole-cell extract.