Figure 1
Figure 1. MLL-BP and MLL fusion proteins downregulate RUNX1 and CBFβ. (A) Schematic diagram demonstrating full-length MLL, MLL-BP (the N-terminal 1406 aa of MLL that is the common part of MLL fusions), and 3 of the most common MLL fusion proteins found in AML patients (MLL-AF9, MLL-ENL, MLL-AF4) used in this study. Immunoblot of RUNX1 (B), CBFβ (C), or RUNX1+CBFβ (D) transiently coexpressed with blank vector, full-length MLL, MLL-BP, MLL-AF9, MLL-ENL, or MLL-AF4 in 293T cells. (E) Human leukemia cell lines were separated into 3 groups: group A, M0-M2 subtypes; group B, M4-M5 subtypes without MLL translocations; and group C, M4-M5 subtypes with MLL translocations. Endogenous RUNX1, CBFβ, and Menin protein expression in these myeloid leukemia cell lines were determined by western blotting. (F) Summary of relative RUNX1, CBFβ, and Menin mRNA levels in the cell lines shown in panel E, which were measured by quantitative real-time polymerase chain reaction and normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) levels using the Δ−CT method. (G) U937 cells were infected with recombinant amphotropic retroviruses carrying either MSCV-PGK-eGFP vector alone or with MLL-BP/MLL-AF9. eGFP+ cells were sorted and lysed for western blotting. (H) Endogenous RUNX1 and CBFβ protein expression in human umbilical cord blood CD34+ cells with Tet-off–driven MLL-AF9 expression. (I) Colony formation of hematopoietic progenitors in wild-type BM cells infected with empty, Meis1, MLL-BP, or MLL-AF9–expressing retroviruses (*P < .05; ***P < .001). The representative image of colonies and cytospin of the first round of plating from Meis1-, MLL-BP–, or MLL-AF9–infected BM cells were shown in the lower panel. (J) Endogenous RUNX1 and CBFβ protein expression in the first round of plating in panel I. (K) Immunoblot of RUNX1 and CBFβ, which were transiently coexpressed with different ratios of MLL and MLL-AF9 in 293T cells. Blank vector was added to make sure 2 μg of total plasmids were transfected in each sample. All of these experiments were repeated 3 times and representative data are shown. DOX, doxycycline; h, hours.

MLL-BP and MLL fusion proteins downregulate RUNX1 and CBFβ. (A) Schematic diagram demonstrating full-length MLL, MLL-BP (the N-terminal 1406 aa of MLL that is the common part of MLL fusions), and 3 of the most common MLL fusion proteins found in AML patients (MLL-AF9, MLL-ENL, MLL-AF4) used in this study. Immunoblot of RUNX1 (B), CBFβ (C), or RUNX1+CBFβ (D) transiently coexpressed with blank vector, full-length MLL, MLL-BP, MLL-AF9, MLL-ENL, or MLL-AF4 in 293T cells. (E) Human leukemia cell lines were separated into 3 groups: group A, M0-M2 subtypes; group B, M4-M5 subtypes without MLL translocations; and group C, M4-M5 subtypes with MLL translocations. Endogenous RUNX1, CBFβ, and Menin protein expression in these myeloid leukemia cell lines were determined by western blotting. (F) Summary of relative RUNX1, CBFβ, and Menin mRNA levels in the cell lines shown in panel E, which were measured by quantitative real-time polymerase chain reaction and normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) levels using the Δ−CT method. (G) U937 cells were infected with recombinant amphotropic retroviruses carrying either MSCV-PGK-eGFP vector alone or with MLL-BP/MLL-AF9. eGFP+ cells were sorted and lysed for western blotting. (H) Endogenous RUNX1 and CBFβ protein expression in human umbilical cord blood CD34+ cells with Tet-off–driven MLL-AF9 expression. (I) Colony formation of hematopoietic progenitors in wild-type BM cells infected with empty, Meis1, MLL-BP, or MLL-AF9–expressing retroviruses (*P < .05; ***P < .001). The representative image of colonies and cytospin of the first round of plating from Meis1-, MLL-BP–, or MLL-AF9–infected BM cells were shown in the lower panel. (J) Endogenous RUNX1 and CBFβ protein expression in the first round of plating in panel I. (K) Immunoblot of RUNX1 and CBFβ, which were transiently coexpressed with different ratios of MLL and MLL-AF9 in 293T cells. Blank vector was added to make sure 2 μg of total plasmids were transfected in each sample. All of these experiments were repeated 3 times and representative data are shown. DOX, doxycycline; h, hours.

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