(E) Cell numbers were counted at indicated days after infection with CD244-targeting shCD244#2 or scrambled shRNA in a representative experiment (n=3). retroviruses were produced in 293T cells with an MSCV-MLL-AF9-IRES-YFP encoding plasmid.16 Lin? fetal liver cells were isolated from wild-type (WT) and CD244 knockout (KO) mice and infected with MLL-AF9 retroviruses by two rounds of spinoculation in the presence of 4 g/mL polybrene. Infected cells (200,000) were transplanted into lethally irradiated (1000 rad) C57BL/6 mice by retro-orbital injection. Indicated YFP+ bone marrow cells Epertinib from primary Epertinib transplanted mice were further infused into recipient mice for secondary transplantation or limiting dilution analysis. Flow cytometry and cell cycle analyses were performed as we described previously. 1 For analysis of lineages and LICs, either peripheral blood or bone marrow cells were stained with anti-mouse Mac-1-APC, anti-mouse Gr-1-PE, anti-mouse CD3-APC, anti-mouse B220-PE, or anti-mouse c-Kit-PE antibodies (eBioscience). CD244 expression on human LICs or HSCs were labeled by the antibodies against human CD45 (FITC), Lineage marker (APC), CD34 (eFluor 450), CD38 (PE-Cy7), CD90 (PE-Cy5.5), CD45RA (PE), CD123 (PE) and CD244 (Biotin). Expression of CD244 and c-Kit on mouse or human LICs (or cell lines) were detected by anti-mouse CD244-PE or human CD244-APC and anti-human c-Kit-PE antibodies (eBioscience). Cell cycle status was measured with Ki-67/7-AAD (or Hoechst 33342) staining (BD Pharmingen) according to the manufacturers instructions. Study approval Bone marrow mononuclear cells were obtained from the patients following diagnostic work at the Department of Hematology at Xinhua Hospital or the 1st Peoples Hospital; human cord blood were obtained from the Department of Gynaecology and Obstetrics at the 6th Peoples Hospital, Shanghai Jiao Tong University School of Medicine. Written informed consent was obtained from all of the patients and all the procedures were approved by the Ethics Committee for Medical Research (IRB) at Shanghai Jiao Tong University School of Medicine. Methods related to mouse information, western blotting and co-immunoprecipitation, quantitative RT-PCR, colony forming unit assays, bone marrow transplantation and statistical analyses are available in the (mean fluorescence intensity) and Physique 1A (frequencies of SIM+ cells), we found that several immune receptors, including IREM-1, CD244 and JAM3, were highly expressed on both human CD34+ hematopoietic stem/progenitor cells (HSPCs) and AML cells transduced with MLL-AF9 oncogene in human CD34+ HSCs, MA9 cells.18 Intriguingly, one of these molecules, CD244, which is critical for functions of NK cells, was expressed at the highest level on both HSPCs and AML cells. To elucidate the roles of CD244 in human AML, we first examined the protein levels of CD244 on different human AML cell lines. Most of the AML cell lines expressed CD244, including Kasumi-1 (M2), NB4 (M3), HL-60 (M3), THP-1 (M5), U937 (M5), MV4-11 (M5) and HEL (M6) (Physique 1B). We then constructed several shRNAs to specifically knockdown CD244 to evaluate its roles in cell proliferation. shCD244#1 and shCD244#2 efficiently reduced the CD244 levels as compared with that in scrambled cells, respectively, measured Rabbit Polyclonal to OR2G2 by either flow cytometric analysis or quantitative RT-PCR (Physique 1C and D). Since shCD244#2 had the highest knockdown efficiency, it was mainly used for the following experiments to unravel the functions of CD244 in human leukemia cells. Open in a separate window Physique 1. CD244 is required for the proliferation of both human leukemia cell lines and Epertinib acute myeloid leukemia initiating cells (LICs). (A) Frequencies of representative surface immune molecules on human cord blood hematopoietic stem/progenitor cells (HSPCs) and MLL-AF9-transduced human MA9 cells were measured by flow cytometric analysis. Cord blood mononuclear cells (MNCs) were used for serving as the control of total cell population. (B) Representative flow cytometric analysis of CD244 expression on different leukemia cell lines including Kasumi-1 (M2), NB4 (M3), HL-60 (M3), THP-1 (M5), U937 (M5), MV4-11 (M5) and HEL (M6) (isotype control, red line). (C and D) Knockdown efficiency of CD244 targeted by scrambled shRNA (Scr), shCD244#1 and shCD244#2 was evaluated by either flow cytometric analysis (C) or quantitative real-time RT-PCR (qRT-PCR) as in (D). (E) Cell numbers were counted at indicated days after contamination with CD244-targeting shCD244#2 or scrambled shRNA in a representative experiment (n=3). (F) MV4-11 cells infected with CD244-targeting shCD244#2 and control cells were injected into NOD-SCID mice. Three months later, engrafted leukemia cells in the bone marrow were detected by anti-human CD45 antibodies (n=6C8). (G and H) Representative images or colony numbers of patients CD34+ AML cells were examined after contamination with CD244-targeting shCD244#2 or scrambled shRNA.