![The nonproteolytic protein ubiquitination network in ALCL. (A) Distribution of mutations in genes related to the inflammatory pathways of ALK−ALCL cell lines, obtained from RNA-seq analysis. (B) A20 protein expression in indicated ALCL lines. (C) TNFAIP3 (A20) mRNA expression in 417 T-cell lymphoma samples (144 peripheral T-cell lymphomas [PTCL] NOS, 127 AITL, 69 ALK− ALCL, 56 ALK+ ALCL, 21 ATLL) and 61 T cells from healthy donors from publicly available microarray data set. t tests were performed between the control normal T-cell group and the other PTCL subtypes, and a P < .05 is considered statistically significant. (D) Overall survival in ALK− ALCL with higher or lower expression values of A20. The threshold (cut-point value for high or low-expression group) was set using maximally selected rank statistics with the “survminer” R package. The cut-point value has been determined as 8.29134 (log2 expression), and the number of patients in the high-expression and low-expression groups is 29 and 8, respectively. (E) NF-κB–driven luciferase reporter–engineered MAC1 line was transduced with inducible lentiviral constructs encoding A20 WT, C103A, and C779/782A complementary DNAs or empty control. Relative NF-κB reporter activities were measured after 1 day of induction. (F) NF-κB–driven luciferase reporter–engineered MAC1 line was transduced with indicated sgRNAs. Relative NF-κB reporter activities were measured after 4 days of induction. (G) MAC1 cells were transduced with RNF31 or ctrl sgRNAs, selected, and expression induced. Cell lysates were subjected to biotin-labeled M1-specific TUBE binding and streptavidin purification and analyzed by immunoblotting. (H) MAC1 cells were transduced with RNF31 or ctrl sgRNAs, selected, and expression induced. IRAK1 immunoprecipitations or total lysates from these lines were immunoblotted for the indicated proteins. In panels E-F, error bars denote SEM of triplicates. P was calculated comparing sgCTRL and the indicated sgRNA groups or comparing empty vector and A20 expression groups; ∗∗P < .01.](https://ash.silverchair-cdn.com/ash/content_public/journal/blood/142/15/10.1182_blood.2022019166/3/blood_bld-2022-019166-gr4.jpeg?Expires=1738083553&Signature=2OB9JZQ2P~IapiptDl27mphKaggujn7nF0xMP3bo1G5IBjXwgPXmok99MjPl8YFCSH3QNOP9CToO~fU5~QctrmMaNskLRPQlGu5maBQTKJqDuqwVBQVJG6XWsInkE0R6iGZJJ8n6gvXzibuWAkipeaNEJenql5THolH3OPySatH1Bw1z9-WGs1CflNhG8zCv2KmK5r9IxUiAAN8-WNNAM09KA2gLzueDczFAhEPaQXY8J8nzLUG4dk6-T2qkl7KBAT7hS-ERdwYJsI1TDWzy2NyltKYv1Fs-J6ZJWx-Jr~PVfv4N61NLP7XlFfHD4yRObBXrWzpUIBKFderwnOnBeg__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
The nonproteolytic protein ubiquitination network in ALCL. (A) Distribution of mutations in genes related to the inflammatory pathways of ALK−ALCL cell lines, obtained from RNA-seq analysis. (B) A20 protein expression in indicated ALCL lines. (C) TNFAIP3 (A20) mRNA expression in 417 T-cell lymphoma samples (144 peripheral T-cell lymphomas [PTCL] NOS, 127 AITL, 69 ALK− ALCL, 56 ALK+ ALCL, 21 ATLL) and 61 T cells from healthy donors from publicly available microarray data set. t tests were performed between the control normal T-cell group and the other PTCL subtypes, and a P < .05 is considered statistically significant. (D) Overall survival in ALK− ALCL with higher or lower expression values of A20. The threshold (cut-point value for high or low-expression group) was set using maximally selected rank statistics with the “survminer” R package. The cut-point value has been determined as 8.29134 (log2 expression), and the number of patients in the high-expression and low-expression groups is 29 and 8, respectively. (E) NF-κB–driven luciferase reporter–engineered MAC1 line was transduced with inducible lentiviral constructs encoding A20 WT, C103A, and C779/782A complementary DNAs or empty control. Relative NF-κB reporter activities were measured after 1 day of induction. (F) NF-κB–driven luciferase reporter–engineered MAC1 line was transduced with indicated sgRNAs. Relative NF-κB reporter activities were measured after 4 days of induction. (G) MAC1 cells were transduced with RNF31 or ctrl sgRNAs, selected, and expression induced. Cell lysates were subjected to biotin-labeled M1-specific TUBE binding and streptavidin purification and analyzed by immunoblotting. (H) MAC1 cells were transduced with RNF31 or ctrl sgRNAs, selected, and expression induced. IRAK1 immunoprecipitations or total lysates from these lines were immunoblotted for the indicated proteins. In panels E-F, error bars denote SEM of triplicates. P was calculated comparing sgCTRL and the indicated sgRNA groups or comparing empty vector and A20 expression groups; ∗∗P < .01.
