Panel A shows a flowchart illustrating the dual purification strategy for mass spectrometry analysis. It includes steps like dual purification, urea treatment, imidazole elution, and dynabeads streptavidin process. Panel B presents a dot plot comparing the top 20 interactors of PML fused to RARA-BioID versus NLS-BioID, highlighting significant SUMO2 conjugation in HPC7 cells. The plot uses fold change and FDR values to indicate the significance of interactions. Panel C displays western blot validations of lysates and purified fractions from HPC7 cells expressing PML fused to RARA-BioID with or without His10-SUMO2, treated with RA and biotin. It shows the presence of various proteins like NCoR, RXRA, KAP1, and ACTIN. Panel D features a gene set enrichment analysis (GSEA) plot showing the enrichment of the PML_BODY pathway in proteomic data. Panel E shows western blot analysis of His10-SUMO3 knock-in mice expressing PML fused to RARA at different stages, highlighting proteins like NCoR, GPS2, IRF2BP2, and TUBULIN. Panel F presents western blot analysis of 293T cells transfected with His10-SUMO2 and various RAR fusion constructs, focusing on NCoR and other proteins. Panel G shows western blot analysis of 293T cells transfected with His10-SUMO2 and PML fused to RARA mutants, highlighting proteins like NCoR, KAP1, GPS2, and RARA. Panel H displays a line graph showing colony counts of Lin- progenitors transformed with RARA and PML fused to RARA mutants at different replating stages, indicating statistical significance with two-way ANOVA.
Fusion of PML to RARA promotes SUMO2 conjugation of RARA-associated partners. (A) Experimental scheme of Ni-NTA plus streptavidin double-purification strategy used for mass spectrometry analysis of HPC7 cells expressing His10-SUMO2 together with NLS-BioID or PML::RARA-BioID. (B) Dot plot of the top 20 PML::RARA-BioID (N = 4) interactors compared to NLS-BioID (N = 4), with significant SUMO2 conjugation in His10-SUMO2–expressing HPC7 cells. (C) Western blot validation of lysates and Ni-NTA– plus streptavidin-purified fractions from HPC7-expressing PML::RARA-BioID alone, or co-expressing His10-SUMO2 with NLS-BioID or PML::RARA-BioID. Cells were pretreated with or without RA (1 μM) for 2 h, followed by biotin (50 μM) addition, with RA treatment continued for a total of 18 h. N = 3. (D) GSEA of proteomics data from His10-SUMO2 HPC7 cells expressing PML::RARA-BioID versus NLS-BioID reveals PML_BODY as the sole significantly enriched pathway. (E) Western blot analysis of His10-SUMO3 knock-in mice expressing PML::RARA at nonleukemic stage (N = 4) or full-blown APL blasts (N = 6) following Ni-NTA purification. (F) Western blot analysis (NCoR antibody) of lysates and Ni-NTA–purified fractions from 293T cells transfected with His10-SUMO2 and indicated RAR fusion constructs. N = 2. (G) Western blot analysis of lysates and Ni-NTA– plus streptavidin double-purified fractions from 293T cells transfected with His10-SUMO2 and the indicated PML::RARA mutants. N = 2. (H) Colony counts of Lin− progenitors transformed with RARA and PML::RARA mutants at the indicated replating. Cells were replated every 5–7 days; MC II, III, and IV denote the second, third, and fourth methylcellulose replating. N = 2. Data are presented as the mean ± SD of three technical triplicates from one representative experiment out of at least two biologically independent experiments. Statistical significance was assessed by two-way ANOVA, ***P < 0.001. Source data are available for this figure: SourceData F3.