Figure 2.
Ythdc1 is required for AML development and maintenance in vivo. (A) Semi-quantitative PCR analysis of Cre and the deletion of Ythdc1, loxP-flanked Ythdc1 allele (loxP), and wild-type (wt) among genomic DNA in BM Lin– cells from Ythdc1fl/+ (WT), Ythdc1fl/+Mx1-Cre (Ythdc1 HET), and Ythdc1fl/flMx1-Cre (Ythdc1 KO) mice; deletion was induced by pIpC injection. Growth curve of Lin– BM cells from WT, Ythdc1 HET, and Ythdc1 KO mice expressed MLL-AF9 (B), AML1-ETO9a (C), PML-Rara (D), and HOXA9 (E). Colony-forming units of Lin– BM cells from WT, Ythdc1 HET, and Ythdc1 KO mice expressed MLL-AF9 (F); representative images of the colonies for the third round (WT and HET) or first round (KO) of plating are displayed (G). Cells were resuspended and replated weekly in MethoCult medium (STEMCELL Technologies) containing cytokines; scale bar, 100 μM. (H) Flow cytometric analysis of engraftment of yellow fluorescent protein (YFP)-positive cells in PB from MLL-AF9-WT (WT), MLL-AF9-Ythdc1 HET (HET), and MLL-AF9-Ythdc1 KO (KO) mice 1 month post-transplantation; n = 5 mice for each group. (I) Complete blood count analysis of WBCs in MLL-AF9-WT (WT), MLL-AF9-Ythdc1 HET (HET), and MLL-AF9-Ythdc1 KO (KO) mice 1 month post-transplantation; n = 5 mice for each group. (J) Kaplan-Meier survival analysis of MLL-AF9-WT (WT), MLL-AF9-Ythdc1 HET (HET), and MLL-AF9-Ythdc1 KO (KO) mice; n = 10 mice for the WT group, n = 8 mice for the HET group, n = 5 mice for the KO group. (K) Wright-Giemsa–stained PB and hematoxylin and eosin–stained spleen and liver of the MLL-AF9-WT (WT), MLL-AF9-Ythdc1 HET (HET), and MLL-AF9-Ythdc1 KO (KO) mice are shown. Scale bars, 20 μM for PB, 100 μM for spleen and liver. (L) Complete blood count analysis of WBCs in MLL-AF9-Ythdc1fl/fl ER Cre mice with or without TAM treatment. Mice were injected with 2 mg of TAM in 100 μL corn oil once a day for 5 consecutive days by intraperitoneal injection when the leukemia cell ratio reached up to 10% in PB; n = 5 mice for each group. (M) Flow cytometric analysis of apoptosis frequency of BM cells isolated from MLL-AF9-Ythdc1fl/fl ER Cre mice with or without TAM treatment 1 month postadministration; n = 4 mice for each group. (N) Wright-Giemsa–stained BM and PB and hematoxylin and eosin–stained liver of the MLL-AF9-Ythdc1fl/fl ER Cre mice with or without TAM treatment 1 month postadministration; Scale bars, 10 μM for BM and PB cells, 100 μM for liver sections. (O) Kaplan-Meier survival analysis of MLL-AF9-Ythdc1fl/fl ER Cre mice with or without TAM treatment; n = 10 mice for the non-treatment group, n = 6 mice for the TAM treatment group. Data are presented as mean ± standard deviation; Student t test or log-rank (Mantel-Cox) test for survival curve. *P < .05; **P < .01; ***P < .001.

Ythdc1 is required for AML development and maintenance in vivo. (A) Semi-quantitative PCR analysis of Cre and the deletion of Ythdc1, loxP-flanked Ythdc1 allele (loxP), and wild-type (wt) among genomic DNA in BM Lin– cells from Ythdc1fl/+ (WT), Ythdc1fl/+Mx1-Cre (Ythdc1 HET), and Ythdc1fl/flMx1-Cre (Ythdc1 KO) mice; deletion was induced by pIpC injection. Growth curve of Lin– BM cells from WT, Ythdc1 HET, and Ythdc1 KO mice expressed MLL-AF9 (B), AML1-ETO9a (C), PML-Rara (D), and HOXA9 (E). Colony-forming units of Lin– BM cells from WT, Ythdc1 HET, and Ythdc1 KO mice expressed MLL-AF9 (F); representative images of the colonies for the third round (WT and HET) or first round (KO) of plating are displayed (G). Cells were resuspended and replated weekly in MethoCult medium (STEMCELL Technologies) containing cytokines; scale bar, 100 μM. (H) Flow cytometric analysis of engraftment of yellow fluorescent protein (YFP)-positive cells in PB from MLL-AF9-WT (WT), MLL-AF9-Ythdc1 HET (HET), and MLL-AF9-Ythdc1 KO (KO) mice 1 month post-transplantation; n = 5 mice for each group. (I) Complete blood count analysis of WBCs in MLL-AF9-WT (WT), MLL-AF9-Ythdc1 HET (HET), and MLL-AF9-Ythdc1 KO (KO) mice 1 month post-transplantation; n = 5 mice for each group. (J) Kaplan-Meier survival analysis of MLL-AF9-WT (WT), MLL-AF9-Ythdc1 HET (HET), and MLL-AF9-Ythdc1 KO (KO) mice; n = 10 mice for the WT group, n = 8 mice for the HET group, n = 5 mice for the KO group. (K) Wright-Giemsa–stained PB and hematoxylin and eosin–stained spleen and liver of the MLL-AF9-WT (WT), MLL-AF9-Ythdc1 HET (HET), and MLL-AF9-Ythdc1 KO (KO) mice are shown. Scale bars, 20 μM for PB, 100 μM for spleen and liver. (L) Complete blood count analysis of WBCs in MLL-AF9-Ythdc1fl/fl ER Cre mice with or without TAM treatment. Mice were injected with 2 mg of TAM in 100 μL corn oil once a day for 5 consecutive days by intraperitoneal injection when the leukemia cell ratio reached up to 10% in PB; n = 5 mice for each group. (M) Flow cytometric analysis of apoptosis frequency of BM cells isolated from MLL-AF9-Ythdc1fl/fl ER Cre mice with or without TAM treatment 1 month postadministration; n = 4 mice for each group. (N) Wright-Giemsa–stained BM and PB and hematoxylin and eosin–stained liver of the MLL-AF9-Ythdc1fl/fl ER Cre mice with or without TAM treatment 1 month postadministration; Scale bars, 10 μM for BM and PB cells, 100 μM for liver sections. (O) Kaplan-Meier survival analysis of MLL-AF9-Ythdc1fl/fl ER Cre mice with or without TAM treatment; n = 10 mice for the non-treatment group, n = 6 mice for the TAM treatment group. Data are presented as mean ± standard deviation; Student t test or log-rank (Mantel-Cox) test for survival curve. *P < .05; **P < .01; ***P < .001.

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