Extended Data Fig. 3: Mutant IDH2 cooperates with mutant Srsf2 to generate lethal MDS with proliferative features in vivo.

a, Schematic of BM transplantation model. b, c, Chimerism of CD45.2⁺ cells in the peripheral blood of recipient mice over time (b) (n = 5 per group at 4 weeks; mean percentage ± s.d.; two-way ANOVA with Tukey’s multiple comparison test) and representative flow cytometry data showing the chimerism of CD45.2⁺ versus CD45.1⁺ (top) or GFP⁺ (bottom) cells in peripheral blood at 16 weeks post-transplant (c) (representative results from five recipient mice; the percentages listed represent the percent of cells within live cells). d, Composition of peripheral blood mononuclear cells (PBMNCs) at 28 weeks post-transplant (the number of analysed mice is indicated; mean + s.d.; two-way ANOVA with Tukey’s multiple comparison tests; statistical significances were detected in percentage of CD11b⁺Gr1⁺ cells in IDH2^R140Q + Srsf2^WT versus IDH2^R140Q + Srsf2^P95H and in IDH2^R172K + Srsf2^WT versus IDH2^R172K + Srsf2^P95H). e–h, Blood counts at 20 weeks post-transplant (white blood cells (WBC) (e); haemoglobin (Hb) (f); platelets (PLT) (g); mean corpuscular volume (MCV) (h); the number of analysed mice is indicated; mean ± s.d.; one-way ANOVA with Tukey’s multiple comparison tests). i, Plasma 2HG levels at 20 weeks post-transplant (2HG levels were quantified as previously described⁴⁰; n = 5 per group were randomly selected; mean ± s.d.; one-way ANOVA with Tukey’s multiple comparison test). j, Correlations between plasma 2HG levels and number of GFP⁺ cells in peripheral blood at 24 weeks post-transplant (n = 5 per group; the Pearson correlation coefficient (R²) and P values (two-tailed) were calculated using PRISM 7). k, Colony numbers from serial replating assays of BM cells collected from end-stage mice from Fig. 2b are shown (mean value ± s.d. represented by lines above the box; the number of analysed mice is indicated; two-way ANOVA with Tukey’s multiple comparison test). l, Giemsa staining of IDH2^R140Q Srsf2^P95H double-mutant cells from the sixth plating (scale bar, 10 μm; original magnification, ×400; representative result from 9 biologically independent experiments). m, Immunophenotype of colony cells at the sixth plating. Normal BM cells were used as a control (the percentage listed represent the percent of cells within live cells; representative result from nine recipient mice). n, Cytomorphology of BM mononuclear cells (BMMNCs) from recipient mice at end stage. BM cells from IDH2 single-mutant and IDH2 and Srsf2 double-mutant groups have increased granulocytes. In addition, IDH2 and Srsf2 double-mutant groups had proliferation of monoblastic and monocytic cells as well as dysplastic features such as abnormally segmented neutrophils (black arrow and inset) and binucleated erythroid precursors with irregular nuclear contours (insets) (scale bar, 10 μm; original magnification, ×400; representative results from 3 controls and 9 recipients are shown; number of mice indicated in o–r). o–r, Blood counts at end-stage (WBC (o); Hb (p); PLT (q); MCV (r); the number of analysed mice is indicated; mean ± s.d.; Kruskal–Wallis tests with uncorrected Dunn’s test). s–u, Results from flow cytometry analysis of BM (s) and peripheral blood (t) mature lineages as well as BM haematopoietic stem/progenitor cells (HSPC) from two tibias, two femurs, and two pelvic bones (u) are quantified (LSK, Lineage⁻SCA1⁺KIT⁺; LT-HSC, long-term haematopoietic stem cell (HSC); ST-HSC, short-term HSC; MPP, multi-potent progenitor; LK: Lineage⁻SCA1⁻KIT⁺; CMP, common myeloid progenitor; GMP, granulocyte-monocyte progenitor; MEP, megakaryocyte-erythroid progenitor; the number of analysed mice is indicated; mean + s.d.; two-way ANOVA with Tukey’s multiple comparison test). v, w, Spleen weight at end stage (v; the number of analysed mice is indicated; mean ± s.d.; two-way ANOVA with Tukey’s multiple comparison test) and representative photographs of spleens from recipient mice from v (w; each photograph was taken with an inch ruler). x, Kaplan–Meier survival analysis of serially transplanted recipient mice that were lethally irradiated (n = 5 per group; log-rank (Mantel–Cox) test (two-sided)). *P < 0.05, **P < 0.01, ***P < 0.001.

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