Panel A shows a Western blot comparing UBC9/RARA expression in vector, UBC9/RARA, and UBC9/RARA I396E-transduced lineage-negative progenitors (MC 3), with GAPDH as the loading control. Panel B shows bright-field microscopy images comparing cellular morphology of Promyelocytic Leukemia Protein-Retinoic Acid Receptor Alpha, Retinoic Acid Receptor Alpha, UBC9/Retinoic Acid Receptor Alpha, and UBC9/Retinoic Acid Receptor Alpha I396E cells (MC 6). Panel C shows a line graph comparing Fc epsilon RI-positive cell percentages across MC 2, MC 4, and MC 6 for Retinoic Acid Receptor Alpha, Promyelocytic Leukemia Protein-Retinoic Acid Receptor Alpha, UBC9/Retinoic Acid Receptor Alpha, and UBC9/Retinoic Acid Receptor Alpha I396E. Panel D shows Ni-NTA purification and input Western blots from 293T cells expressing His-SUMO2 with Promyelocytic Leukemia Protein-Retinoic Acid Receptor Alpha and UBC9/Retinoic Acid Receptor Alpha constructs, detecting NCoR, Retinoic Acid Receptor Alpha, GPS2, IRF2BP2, and ACTIN. Panel E shows fluorescence microscopy images of untreated and TAK-981-treated cells expressing Promyelocytic Leukemia Protein-Retinoic Acid Receptor Alpha, Promyelocytic Leukemia Protein-Retinoic Acid Receptor Alpha L278E, Promyelocytic Leukemia Protein-Retinoic Acid Receptor Alpha I396E, Promyelocytic Leukemia Protein-Retinoic Acid Receptor Alpha K160R, UBC9/Retinoic Acid Receptor Alpha, UBC9/Retinoic Acid Receptor Alpha I396E, and Retinoic Acid Receptor Alpha, with red fluorescence and blue nuclear staining.
Exploration of UBC9/RARA and its I396E mutant. (A) Western blot analysis of UBC9/RARA and UBC9-RARAI396E expression in Lin− progenitors at the third methylcellulose passage (MC III). Representative cells for N = 3. (B) MGG staining of primary mouse Lin− hematopoietic progenitors transformed with UBC9/RARA, RARA fusions at the sixth passage (MC VI). Scale bar, 10 μm. N = 3. (C) FACS analysis of mast cell marker FcεRI expression in Lin− progenitors transformed with indicated fusions, at the specified replating. N = 2. (D) Western blot analysis of lysates and Ni-NTA–purified His-tagged SUMO conjugates from Lin− progenitors transduced with indicated constructs. N = 2. PML::RARA-S or UBC9-RARA-S denotes SUMO-conjugated PML::RARA or UBC9/RARA. (E) TAK-981 does not alter the cellular localization of RARA fusions. Confocal analysis of subcellular localization of RARA, PML::RARA, UBC9/RARA, and their mutants in Lin− progenitors after 16 h of TAK-981 (100 nM) treatment in methylcellulose, detected with an anti-hRARA antibody. Representative cells for N = 2. Scale bar, 10 mm. Source data are available for this figure: SourceData FS2.