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Additional Supporting Information may be found in the online version of this article.

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IJC_27904_sm_SuppFig1a.tif140KSupporting Information Figure 1a. Expression of CD34, CD38, and CD82. (a) CD34+/CD38- and CD34+/CD38+ cells isolated from AML patients (case #s 1 and 2) were gated. Expression of CD82 in CD34+/CD38- AML cells and their CD34+/CD38+ counterparts was analyzed using FlowJo. AML cells isolated from patients (case #s 1 and 2) were purified by magnetic cell sorting utilizing a CD34 MultiSort kit and a CD38 MicroBead kit. Expression of CD34 and CD38 was analyzed using FlowJo. Histgram of FACS. (b) Expression of GFP in CD34+/CD38- AML cells transduced with CD82 shRNA was analyzed using FACscan. Expression of V5 in CD34+/CD38+ AML cells transduced with CD82-expressing vector was analyzed using FlowJo.
IJC_27904_sm_SuppFig1b.tif86KSupporting Information Figure 1b. Expression of CD34, CD38, and CD82. (a) CD34+/CD38- and CD34+/CD38+ cells isolated from AML patients (case #s 1 and 2) were gated. Expression of CD82 in CD34+/CD38- AML cells and their CD34+/CD38+ counterparts was analyzed using FlowJo. AML cells isolated from patients (case #s 1 and 2) were purified by magnetic cell sorting utilizing a CD34 MultiSort kit and a CD38 MicroBead kit. Expression of CD34 and CD38 was analyzed using FlowJo. Histgram of FACS. (b) Expression of GFP in CD34+/CD38- AML cells transduced with CD82 shRNA was analyzed using FACscan. Expression of V5 in CD34+/CD38+ AML cells transduced with CD82-expressing vector was analyzed using FlowJo.
IJC_27904_sm_SuppFig1c.tif160KSupporting Information Figure 1c. Expression of CD34, CD38, and CD82. (a) CD34+/CD38- and CD34+/CD38+ cells isolated from AML patients (case #s 1 and 2) were gated. Expression of CD82 in CD34+/CD38- AML cells and their CD34+/CD38+ counterparts was analyzed using FlowJo. AML cells isolated from patients (case #s 1 and 2) were purified by magnetic cell sorting utilizing a CD34 MultiSort kit and a CD38 MicroBead kit. Expression of CD34 and CD38 was analyzed using FlowJo. Histgram of FACS. (b) Expression of GFP in CD34+/CD38- AML cells transduced with CD82 shRNA was analyzed using FACscan. Expression of V5 in CD34+/CD38+ AML cells transduced with CD82-expressing vector was analyzed using FlowJo.
IJC_27904_sm_SuppFig2a.tif388KSupporting Information Figure 2a. Histgram of FACS. (a) CD34+/CD38- and CD34+/CD38+ cells isolated from AML patients (case #s 3, 9, 10 and 14) and (b) CD34+ hematopoietic stem/progenitor cells isolated from healthy volunteers (n=6) were gated. Expression of CD82 in CD34+/CD38- AML cells, their CD34+/CD38+ counterparts, and CD34+ hematopoietic stem/progenitor cells was analyzed using FACscan. (c) Expression of CD82 in EOL-1 and EOL-1R cells was analyzed using FlowJo.
IJC_27904_sm_SuppFig2b.tif105KSupporting Information Figure 2b. Histgram of FACS. (a) CD34+/CD38- and CD34+/CD38+ cells isolated from AML patients (case #s 3, 9, 10 and 14) and (b) CD34+ hematopoietic stem/progenitor cells isolated from healthy volunteers (n=6) were gated. Expression of CD82 in CD34+/CD38- AML cells, their CD34+/CD38+ counterparts, and CD34+ hematopoietic stem/progenitor cells was analyzed using FACscan. (c) Expression of CD82 in EOL-1 and EOL-1R cells was analyzed using FlowJo.
IJC_27904_sm_SuppFig3.tif149KSupporting Information Figure 3. The effect of CD82 on cell cycle of leukemia cells. EOL-1R cells were transfected with either scrambled control or CD82 siRNA. CD34+/CD38- AML cells (case #s 1 and 6) were transduced with either control or CD82 shRNA. The cell cycle distribution of these cells was analyzed by flow cytometry. G0 phase is defined as cells that are negative for expression of nuclear antigen Ki-67 (lower left quadrant). G1 phase; lower left quadrant. S phase; hither right quadrant.
IJC_27904_sm_SuppFig4.tif64KSupporting Information Figure 4. Colony forming assay. The effect of CD82 on proliferation of CD34+ PBSCs isolated from healthy volunteers (n=3). CD34+ cells transduced with CD82 shRNA were cultured in methylcellulose medium. After 14 days, colonies were counted. Each dot represents colony number compared with control for an individual experiment and the mean is indicated by the line.
IJC_27904_sm_SuppFig5.tif92KSupporting Information Figure 5. The link between IL-10 and MMP9 in leukemia cells. CD34+/CD38- AML cells (case #s 2 and 6) transduced with either control or IL-10 shRNA and CD34+/CD38- AML cells (case #s 1 and 6) cultured with IL-10 (5ng/ml) were collected and mRNAs were extracted. cDNAs were synthesized and subjected to real-time RT-PCR to measure the levels of IL-10 and MMP9. Each dot represents the levels of MMP9 for an individual experiment and the mean is indicated by the line. **, p<0.01; *, p<0.05.
IJC_27904_sm_SuppFig6.tif60KSupporting Information Figure 6. The effect of CD82 on BM homing. The human AML cells isolated from three patients (case #s 6, 14, and 17) were treated with anti-CD82 (CD82 mAb) or control IgG (IgG) antibody on ice for 1 h. Cells were injected to each mouse (IgG n=6, CD82 mAb n=6) intravenously via the tail vein. At 16 hrs after transplantation, mice were euthanized and BMs were removed and analyzed by flow cytometry with anti-human CD45 and CD34 antibodies. Each dot represents CD45 and CD34 expression for an individual experiment and the mean is indicated by the line. **, p<0.01; *, p<0.05.
IJC_27904_sm_SuppFig7.tif58KSupporting Information Figure 7. The effect of integrin ?1 on adhesion of AML cells. Real time RT-PCR. (a) RNA was extracted from CD34+/CD38- cells and their CD34+/CD38+ counterparts isolated from AML patients. cDNAs were synthesized and subjected to real-time RT-PCR to measure the levels of VLA4. Each dot represents the levels of VLA4 for an individual experiment and the mean is indicated by the line. Migration assays. (b) CD34+/CD38+ AML cells (case# 15) transduced with CD82-expressing lentiviral particle were treated with anti-integrin ?1 (CD29 mAb) or control IgG (IgG) antibody on ice for 1 h. These cells were seeded in the upper biocoat cell culture inserts. After 48 hrs, the cells that had migrated through the filters were stained with 4′6-diamidino-2-phenylindole, and counted under a microscope. **, p<0.01; *, p<0.05. Immunoprecipitation. (c) EOL-1R cells were harvested and proteins were extracted. The CD82 or IgG protein was immunoprecipitated and subjected to Western blot analysis. The membrane was probed sequentially with an anti-CD82 antibody (bottom) and an anti-integrin α4 (upper).
IJC_27904_sm_SuppFig7b.tif104KSupporting Information Figure 7b. The effect of integrin ?1 on adhesion of AML cells. Real time RT-PCR. (a) RNA was extracted from CD34+/CD38- cells and their CD34+/CD38+ counterparts isolated from AML patients. cDNAs were synthesized and subjected to real-time RT-PCR to measure the levels of VLA4. Each dot represents the levels of VLA4 for an individual experiment and the mean is indicated by the line. Migration assays. (b) CD34+/CD38+ AML cells (case# 15) transduced with CD82-expressing lentiviral particle were treated with anti-integrin ?1 (CD29 mAb) or control IgG (IgG) antibody on ice for 1 h. These cells were seeded in the upper biocoat cell culture inserts. After 48 hrs, the cells that had migrated through the filters were stained with 4′6-diamidino-2-phenylindole, and counted under a microscope. **, p<0.01; *, p<0.05. Immunoprecipitation. (c) EOL-1R cells were harvested and proteins were extracted. The CD82 or IgG protein was immunoprecipitated and subjected to Western blot analysis. The membrane was probed sequentially with an anti-CD82 antibody (bottom) and an anti-integrin α4 (upper).
IJC_27904_sm_SuppTab1.doc51KSupporting Information Table 1.
IJC_27904_sm_SuppTab2.doc141KSupporting Information Table 2.

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