Figure 2.
The leukemia-suppressive functions of TET proteins are preserved in the absence of PROSER1. (A) Box plots showing cell numbers (left) and colony-forming units (CFUs; right) of wild-type, TET2 KO, and PROSER1 KO HOXB8-FL cells grown in semisolid media (+E2 and +Flt3L) for 7 days. Each dot represents an independently derived cell line (n = 4). ∗∗P < .01; ∗∗∗∗P < .0001 (Brown-Forsythe and Welch analysis of variance [ANOVA] test). (B) Same as in panel A but for HOXB8-FL cells expressing AE onco-fusion protein and replated 5 times (7 days each) in semisolid media without E2 and with cytokines (+stem cell factor, +interleukin-3 [IL-3], and +IL-6) to support leukemic blast cell growth. ∗P < .05 (unpaired 2-tailed t test with individual variance for each replating). (C) Schematic illustrating AE in vivo leukemia transplantation assay. (D) Kaplan-Meier plot showing disease-free survival of recipient mice transplanted with AE expressing HOXB8-FL cells of the indicated genotypes. ∗∗∗P < .001 (Log-rank [Mantel-Cox] test). (E) Proportion of GFP-positive AE-expressing leukemic blasts in BM of recipient mice at the time of harvest. ∗∗P < .01 (unpaired 2-tailed t test with Welch correction). (F) Box plots showing cell numbers (left) and CFUs (right) of wild-type, TET2 KO, and PROSER1/TET2 DKO HOXB8-FL cells grown in semisolid media (+E2 and +Flt3L) for 7 days. Each dot represents an independently derived cell line (n = 4). ∗∗∗P < .001; ∗∗∗∗P < .0001 (Brown-Forsythe and Welch ANOVA test). (G) Schematic illustrating B-cell malignancy transplantation assay. (H) Representative fluorescence-activated cell sorter (FACS) plots showing expansion of immature B-cell blasts in BM 12 weeks after transplantation of TET2/TET3 DKO HOXB8-FL cells. Immature B-cell blasts are not found in mice receiving HOXB8-FL cells with PROSER1/TET2 DKO as well as PROSER1 KO, TET2 KO, and TET3 KO. (I) Box plot showing absolute counts of CD45.2+B220+cKit+ immature B-cell blasts in BM at 12 weeks after transplantation into lethally irradiated CD45.1 recipient mice. ∗∗P < .01 (unpaired 2-tailed t test with Welch correction). GFP, green fluorescent protein; ns, nonsignificant; p1, plating no.; WT, wild-type.

The leukemia-suppressive functions of TET proteins are preserved in the absence of PROSER1. (A) Box plots showing cell numbers (left) and colony-forming units (CFUs; right) of wild-type, TET2 KO, and PROSER1 KO HOXB8-FL cells grown in semisolid media (+E2 and +Flt3L) for 7 days. Each dot represents an independently derived cell line (n = 4). ∗∗P < .01; ∗∗∗∗P < .0001 (Brown-Forsythe and Welch analysis of variance [ANOVA] test). (B) Same as in panel A but for HOXB8-FL cells expressing AE onco-fusion protein and replated 5 times (7 days each) in semisolid media without E2 and with cytokines (+stem cell factor, +interleukin-3 [IL-3], and +IL-6) to support leukemic blast cell growth. ∗P < .05 (unpaired 2-tailed t test with individual variance for each replating). (C) Schematic illustrating AE in vivo leukemia transplantation assay. (D) Kaplan-Meier plot showing disease-free survival of recipient mice transplanted with AE expressing HOXB8-FL cells of the indicated genotypes. ∗∗∗P < .001 (Log-rank [Mantel-Cox] test). (E) Proportion of GFP-positive AE-expressing leukemic blasts in BM of recipient mice at the time of harvest. ∗∗P < .01 (unpaired 2-tailed t test with Welch correction). (F) Box plots showing cell numbers (left) and CFUs (right) of wild-type, TET2 KO, and PROSER1/TET2 DKO HOXB8-FL cells grown in semisolid media (+E2 and +Flt3L) for 7 days. Each dot represents an independently derived cell line (n = 4). ∗∗∗P < .001; ∗∗∗∗P < .0001 (Brown-Forsythe and Welch ANOVA test). (G) Schematic illustrating B-cell malignancy transplantation assay. (H) Representative fluorescence-activated cell sorter (FACS) plots showing expansion of immature B-cell blasts in BM 12 weeks after transplantation of TET2/TET3 DKO HOXB8-FL cells. Immature B-cell blasts are not found in mice receiving HOXB8-FL cells with PROSER1/TET2 DKO as well as PROSER1 KO, TET2 KO, and TET3 KO. (I) Box plot showing absolute counts of CD45.2+B220+cKit+ immature B-cell blasts in BM at 12 weeks after transplantation into lethally irradiated CD45.1 recipient mice. ∗∗P < .01 (unpaired 2-tailed t test with Welch correction). GFP, green fluorescent protein; ns, nonsignificant; p1, plating no.; WT, wild-type.

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