Extended Data Fig. 4: Collaborative effects of mutant Idh2 and mutant Srsf2 are not dependent on Tet2 loss alone.

a, Schematic of competitive and non-competitive transplantation assays of CD45.2⁺ Mx1-cre control, Mx1-cre Idh2^R140Q/+, Mx1-cre Srsf2^P95H/+, Mx1-cre Idh2^R140Q/+Srsf2^P95H/+ mice, Mx1-cre Tet2^fl/fl, Mx1-cre Tet2^fl/flSrsf2^P95H/+ mice into CD45.1⁺ recipient mice. b, 2HG levels of bulk PBMNCs from primary Mx1-cre mice were measured at three months post-pIpC (polyinosinic:polycytidylic acid) and normalized to internal standard (d-2-hydroxyglutaric-2,3,3,4,4-d5 acid; D5-2HG) (2HG and D5-2HG levels were quantified as described⁴⁰; n = 5 per group; mean ± s.d.; one-way ANOVA with Tukey’s multiple comparison test). c, DNA extracted from sorted KIT⁺ BM cells from primary Mx1-cre mice at one month post-pIpC was probed with antibodies specific for 5-hydroxymethylcytosine (5hmC) (left). Relative intensity of each dot was measured by ImageJ and divided by input DNA amount for comparison (right; n = 4; intensity of each dot divided by amount of input DNA was combined per genotype; representative results from 3 biologically independent experiments with similar results; mean ± s.d.; one-way ANOVA with Tukey’s multiple comparison test). d, Chimerism of peripheral blood CD45.2⁺ cells in non-competitive transplantation (pIpC was injected at 4 weeks post-transplant; mean ± s.d.; n = 10 (control and Idh2^R140Q), n = 8 (Srsf2^P95H), and n = 9 (DKI) at 0 week; two-way ANOVA with Tukey’s multiple comparison test; P values from comparison between Srsf2^P95H and each of other groups are shown). e–i, Absolute number of BM HSPCs from two tibias, two femurs, and two pelvic bones were measured in the primary (e, f) and serial (h, i) competitive transplant of Idh2 and Srs2 mutant cells, and representative flow cytometry of BM HSPCs from the primary competitive transplant of Idh2 and Srsf2 mutant cells from e, f (the percentage listed represents the percent of cells within live cells) (the number of analysed mice is indicated; mean + s.d.; two-way ANOVA with Tukey’s multiple comparison test). j, Kaplan–Meier survival analysis of CD45.1⁺ recipient mice transplanted non-competitively with BM cells from CD45.2⁺ Mx1-cre control, Mx1-cre Tet2^fl/fl, Mx1-cre Srsf2^P95H/+, and Mx1-cre Tet2^fl/flSrsf2^P95H/+ mice (pIpC was injected at 4 weeks post-transplant; n = 10 per genotype; log-rank (Mantel–Cox) test (two-sided)). k, l, Chimerism of peripheral blood CD45.2⁺ cells in non-competitive (k) (n = 10 (control and Tet2 knockout (Tet2KO)), n = 8 (Srsf2^P95H), and n = 5 (Tet2KO + Srsf2^P95H) at 0 weeks) or competitive (l) (n = 9 (control), n = 10 (Tet2KO), n = 8 (Srsf2^P95H), and n = 10 (Tet2KO + Srsf2^P95H) at 0 weeks) transplantation (pIpC was injected at 4 weeks post-transplant; percentages of CD45.2⁺ cells at pre-transplant are also shown as data at 0 weeks in l; mean ± s.d.; two-way ANOVA with Tukey’s multiple comparison test). m, n, Absolute number of BM HSPCs from two tibias, two femurs, and two pelvic bones were measured in the primary competitive transplant of Tet2 and Srsf2 mutant cells (n = 10 per genotype; mean + s.d.; two-way ANOVA with Tukey’s multiple comparison test). o, Schematic of TET2 catalytic domain (CD: catalytic domain; EV: empty vector) retroviral BM transplantation model. p, Western blot analysis confirming the expression of Myc-tagged TET2 CD in Ba/F3 cells transduced with or without TET2 CD (representative images from two biologically independent experiments with similar results). q, Chimerism of mCherry–TET2 CD⁺ and GFP–EV⁺ cells in peripheral blood of recipient mice over time (n = 10; mean percentage ± s.d.; two-way ANOVA with Sidak’s multiple comparison test). r, qPCR of Tet3 in the first colony cells from s (n = 3; mean ± s.d.; a two-sided Student’s t-test). s, Colony numbers from serial replating assays of BM cells from Mx1-cre control, Mx1-cre Srsf2^P95H/+, and Mx1-cre Tet2^fl/flSrsf2^P95H/+ mice transduced with shRNAs targeting Tet3 (shTet3) (n = 3; mean + s.d.; two-way ANOVA with Tukey’s multiple comparison test). t, Schematic of shTet3 retroviral BM transplantation model. u, v, Chimerism of mCherry⁺ cells in CD45.2⁺ donor cells in peripheral blood of recipient mice over time (u; left, Mx1-cre Srsf2^P95H/+; right, Mx1-cre Tet2^fl/flSrsf2^P95H/+; n = 5 per group) and at 20 weeks post-transplant (v) (mean percentage ± s.d.; two-way ANOVA with Sidak’s multiple comparison test). w, Colony numbers from serial replating assays of either Mx1-cre Srsf2^+/+ or Srsf2^P95H/+ BM cells transduced with an shRNA against Fto or Alkbh5. BM cells were collected at one month post-pIpC (n = 3; mean value ± s.d.; two-way ANOVA with Tukey’s multiple comparison test). x, qPCR of Fto or Alkbh5 in Ba/F3 cells transduced with shRNAs targeting mouse Fto or Alkbh5 (n = 3; mean value ± s.d.; one-way ANOVA with Tukey’s multiple comparison test). *P < 0.05, **P < 0.01, ***P < 0.001.

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