Fig. 2: Characterization of RUNX1::ERG and STIM1::MXD3.

a Schematic diagrams showing RUNX1, ERG, and RUNX1::ERG. b Localization of RUNX1::ERG (R::E), STIM1::MXD3 (S::M) and the wild-type counterparts in transfected HeLa cells. Tagged proteins were detected with an anti-HA antibody. Original magnification ×1000. Scale bars: 10 µm. c Transcriptional properties of RUNX1::ERG. Promoter constructs were co-transfected with the indicated expression plasmids into 293T cells. EV empty vector. d RUNX1::ERG inhibited RUNX1- and ERG-mediated transcriptional activation. Promoter constructs were co-transfected with the indicated expression plasmids in the presence of increasing amounts of pCMV-HA-RUNX1::ERG. Fold activation was compared to parallel transfections using the same amount of pCMV-HA empty vector. e MYC repression by RUNX1::ERG. GSEA comparing the diagnostic and CD34⁺-enriched remission bone marrow samples (both ≥90% of CD34 positivity) of the patient with RUNX1::ERG as well as K562 cells transfected with LeGO-iG2-RUNX1::ERG or the empty LeGO-iG2. For both patient and cell line samples, two biological replicates from each group were analysed. f Western blotting showing MYC repression in K562 cells transfected with LeGO-iG2-RUNX1::ERG (R::E) or the empty LeGO-iG2 (EV). GAPDH served as the loading control. MYC and GAPDH were probed from the same blot, while R::E was from a different blot. The results of the densitometry analysis are shown below the blot images. g Effects of RUNX1::ERG overexpression on transfected K562 cells. Cell size (indicated by the median fluorescence intensity of forward size scatter (FSC)), percentage of proliferating cells, and cell cycle were determined by flow cytometry. h RUNX1::ERG overexpression inhibited colony formation of CD34⁺ hematopoietic stem/progenitor cells. i Transcriptional properties of STIM1::MXD3. The Myc reporter construct was co-transfected with the indicated expression plasmids into 293T cells. j GSEA comparing K562 cells overexpressing STIM1::MXD3 or MXD3 with the empty vector control, and patient samples with NUP98::NSD1⁺/STIM1::MXD3⁺ (n = 1) or NUP98::NSD1⁺/STIM1::MXD3^-/STIM1::F12^- (n = 2). Two biological replicates of each K562 transfection were analysed. No significant MYC enrichment was noted between the MXD3 and empty vector transfection comparison. Data in charts are expressed as mean ± SE from three to four independent experiments. The number of values used to calculate the statistics (one-way ANOVA followed by Dunnett’s test in c, d, i, and t-test in f, g, h) in each group is indicated. The hallmark gene sets were used for GSEA in e, j.