![Epigenetic change in part governs B-myeloid conversion. (A) Western blot of NF-κB (right) and Notch (left) signaling proteins in TriB splenic B cells (TriB), normal splenic B cells (Con), converted myeloid leukemia cells (Bswitch), and transformed TriB lymphoma cells (TriB[lym]). Actin is used as a loading control. Note that these results further confirm the activation of both NF-κB and Notch signaling in TriB mice. (B) Genomic PCR analysis of Cre-mediated transgene recombination in converted myeloid leukemia cells (Bswitch) from 2 different mice. TriB genomic DNA was used for detection of transgenes without recombination. Note the deletion of transgene Neo gene after Cre-mediated recombination. (C) Quantitative PCR (qPCR) analysis of Gt(ROSA)26Sor expression in TriB MZPs (MZP) and converted myeloid leukemia cells (Bswitch; n = 3). (D) Chromatin immunoprecipitation (ChIP)–qPCR measurement of histone 3 lysine 27 acetylation (H3K27ac) in the promoter regions of Gt(ROSA)26Sor, Itgam (CD11b), and Cd19 genes in TriB MZPs and converted myeloid cells (Bswitch). (E) ATAC-seq signal coverage across Gt(ROSA)26Sor, Itgam, and Cd19 regions in TriB MZPs (upper) and Bswitch cells (lower) were visualized with Integrated Genomics Viewer. (F) Relative expression of Tet2 and Dnmt1 in TriB MZPs (MZP) and converted myeloid leukemia cells (Bswitch) by qPCR (n = 3). (G) Determination of cell growth in vitro indicating that DNA demethylating drug 5-aza-2′-deoxycytidine (5AZA) suppressed the growth of converted myeloid leukemia cells (n = 3). (H) Flow cytometric analysis of GFP expression in converted myeloid leukemia cells treated with 5AZA (n = 3); representative contour plots comparing untreated cells (dimethyl sulfoxide [DMSO]) with treated cells (left), and recovery of GFP expression upon treatment using 5AZA (right). (I) Expression levels of NIK and Notch2ICN (ICN) before and after drug treatment (5AZA) of converted myeloid leukemia cells (n = 3). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is used as a loading control. *P < .05, **P < .01.](https://ash.silverchair-cdn.com/ash/content_public/journal/blood/135/2/10.1182_blood.2019001438/2/bloodbld2019001438f5.png?Expires=1719172497&Signature=sMU4sjsyk1RFQpet3rArurFbnwHgJzGltSYc3u6xGkYWMR3CvJ~uXUWwtOiYryTJQrIcyvS3u~1syCCKV7fhz7H35lmvKM8yOUve2lutAHeOdR5mlIL4104oc3vHgWQznQQABeFuWsua3cZ09Fuia7rANM8gr0L4HJgCKIszOolrc5OSyFVh-VVSZ9X6R1d8w1pVQpqICoXe6M6QZYqMZgI~oWylUSlWyb7QihEh1YGCgXe3BVoxktmoRd-SucpRsSwMIYlzWd4~jXa-GiEh~lhf4wfF3Scy7vAfrip7ZF4Kno10hyuF1zDx8FLbnWWvlxtUSqTiPxCMy75cg9CeoA__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
Epigenetic change in part governs B-myeloid conversion. (A) Western blot of NF-κB (right) and Notch (left) signaling proteins in TriB splenic B cells (TriB), normal splenic B cells (Con), converted myeloid leukemia cells (Bswitch), and transformed TriB lymphoma cells (TriB[lym]). Actin is used as a loading control. Note that these results further confirm the activation of both NF-κB and Notch signaling in TriB mice. (B) Genomic PCR analysis of Cre-mediated transgene recombination in converted myeloid leukemia cells (Bswitch) from 2 different mice. TriB genomic DNA was used for detection of transgenes without recombination. Note the deletion of transgene Neo gene after Cre-mediated recombination. (C) Quantitative PCR (qPCR) analysis of Gt(ROSA)26Sor expression in TriB MZPs (MZP) and converted myeloid leukemia cells (Bswitch; n = 3). (D) Chromatin immunoprecipitation (ChIP)–qPCR measurement of histone 3 lysine 27 acetylation (H3K27ac) in the promoter regions of Gt(ROSA)26Sor, Itgam (CD11b), and Cd19 genes in TriB MZPs and converted myeloid cells (Bswitch). (E) ATAC-seq signal coverage across Gt(ROSA)26Sor, Itgam, and Cd19 regions in TriB MZPs (upper) and Bswitch cells (lower) were visualized with Integrated Genomics Viewer. (F) Relative expression of Tet2 and Dnmt1 in TriB MZPs (MZP) and converted myeloid leukemia cells (Bswitch) by qPCR (n = 3). (G) Determination of cell growth in vitro indicating that DNA demethylating drug 5-aza-2′-deoxycytidine (5AZA) suppressed the growth of converted myeloid leukemia cells (n = 3). (H) Flow cytometric analysis of GFP expression in converted myeloid leukemia cells treated with 5AZA (n = 3); representative contour plots comparing untreated cells (dimethyl sulfoxide [DMSO]) with treated cells (left), and recovery of GFP expression upon treatment using 5AZA (right). (I) Expression levels of NIK and Notch2ICN (ICN) before and after drug treatment (5AZA) of converted myeloid leukemia cells (n = 3). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is used as a loading control. *P < .05, **P < .01.