1. Megakaryocyte-derived excessive transforming growth factor β1 inhibits proliferation of normal hematopoietic stem cells in acute myeloid leukemia 
Yuemin Gong, Mei Zhao, Wanzhu Yang, Ai Gao, Xiuxiu Yin, Linping Hu, Xiaofang Wang, Jing Xu, Sha Hao, Tao Cheng, Hui Cheng 
Experimental Hematology 
Volume 60, Pages e2 (April 2018)
DOI: /j.exphem
Copyright © 2018 ISEH – Society for Hematology and Stem Cells Terms and Conditions

2. Figure 1
Upregulation of TGFβ signaling pathway in HSCs isolated from AML hosts. (A) Experimental design for a nonirradiated MLL-AF9-induced AML mouse model. BM lineage-negative cells infected with MSCV-MLL-AF9-IRES-eGFP viruses were transplanted into lethally irradiated mice to establish the primary AML model. Leukemic marrow infiltration reveals a novel role for Egr3 as a potent inhibitor of normal hematopoietic stem cell proliferation. Leukemic cells were then isolated from spleens of primary AML mice and transplanted into non-irradiated mice to induce secondary AML for subsequent experiments. (B) Percentages of eGFP+ leukemia cells in mouse BM at indicated time points postinjection of leukemia cells. (C) GSEA plots of TGFβ1 target genes (left) and SMAD2/3 pathway (right) in normal LKS+ (Lin–ckit+Sca1+) cells from mice with AML (early and late stages) versus healthy control mice. Both gene sets were downloaded from GSEA Molecular Signatures Database Version 6.0. (D) Expression of genes enriched in TGFβ signaling and with significant upregulation (p < 0.05) in LKS+ cells from mice with AML versus control (Ctrl) mice. (E) Proliferation rates of LKS+ cells after 7 days of incubation in AML versus control BM plasma conditioned medium to which was added SB (0.5 µmol/L) or DMSO. BM plasma was mixed with culture medium (SFEM supplemented with 100 ng/mL rmSCF and 100 ng/mL rhTPO) at a ratio of 1:1 (three independent experiments). Significant differences: *p < 0.05. Error bars represent SD. AML=acute myeloid leukemia; BM=bone marrow; DMSO=dimethyl sulfoxide; eGFP=enhanced green fluorescent protein; GSEA=gene set enrichment analysis; HSCs=hematopoietic stem cells; NES=normalized enrichment score; ns=no significance; p = nominal p value; rhTPO=recombinant human thrombopoietin; rmSCF=recombinant mouse stem cell factor; SFEM=StemCell serum-free expansion medium; TGFβ1=transforming growth factor β1.
Experimental Hematology  , e2DOI: ( /j.exphem )
Copyright © 2018 ISEH – Society for Hematology and Stem Cells Terms and Conditions

3. Figure 1
Upregulation of TGFβ signaling pathway in HSCs isolated from AML hosts. (A) Experimental design for a nonirradiated MLL-AF9-induced AML mouse model. BM lineage-negative cells infected with MSCV-MLL-AF9-IRES-eGFP viruses were transplanted into lethally irradiated mice to establish the primary AML model. Leukemic marrow infiltration reveals a novel role for Egr3 as a potent inhibitor of normal hematopoietic stem cell proliferation. Leukemic cells were then isolated from spleens of primary AML mice and transplanted into non-irradiated mice to induce secondary AML for subsequent experiments. (B) Percentages of eGFP+ leukemia cells in mouse BM at indicated time points postinjection of leukemia cells. (C) GSEA plots of TGFβ1 target genes (left) and SMAD2/3 pathway (right) in normal LKS+ (Lin–ckit+Sca1+) cells from mice with AML (early and late stages) versus healthy control mice. Both gene sets were downloaded from GSEA Molecular Signatures Database Version 6.0. (D) Expression of genes enriched in TGFβ signaling and with significant upregulation (p < 0.05) in LKS+ cells from mice with AML versus control (Ctrl) mice. (E) Proliferation rates of LKS+ cells after 7 days of incubation in AML versus control BM plasma conditioned medium to which was added SB (0.5 µmol/L) or DMSO. BM plasma was mixed with culture medium (SFEM supplemented with 100 ng/mL rmSCF and 100 ng/mL rhTPO) at a ratio of 1:1 (three independent experiments). Significant differences: *p < 0.05. Error bars represent SD. AML=acute myeloid leukemia; BM=bone marrow; DMSO=dimethyl sulfoxide; eGFP=enhanced green fluorescent protein; GSEA=gene set enrichment analysis; HSCs=hematopoietic stem cells; NES=normalized enrichment score; ns=no significance; p = nominal p value; rhTPO=recombinant human thrombopoietin; rmSCF=recombinant mouse stem cell factor; SFEM=StemCell serum-free expansion medium; TGFβ1=transforming growth factor β1.
Experimental Hematology  , e2DOI: ( /j.exphem )
Copyright © 2018 ISEH – Society for Hematology and Stem Cells Terms and Conditions

4. Figure 2
Excessive production of TGFβ1 in AML BM. (A) TGFβ1 concentration in control and AML BM plasma at indicated time points was measured by ELISA (three independent experiments). (B) Expression of Tgfb1 was quantified by qRT-PCR in indicated cell types isolated from AML (day 7) bone marrow compared with control (three independent experiments). (C) Left: Representative FACS plot for measurement of intracellular TGFβ1 protein in megakaryocytes (MKs). Right: Bar plot of TGFβ1 MFI in MKs (three independent experiments). (D) Top: Representative images of MKs in control and AML (day 7) BM Bottom: In situ measurement of TGFβ1 MFI in MKs from AML (n = 40) versus control (n = 43) BM (two independent experiments). (E) Trans-well co-culture of MKs and leukemic blasts (1:10). MKs were derived from c-Kit+ bone marrow cells cultured in vitro with rhTPO (100 ng/mL) for 6 days. Leukemic cells were isolated from secondary AML mice at day 14 post-leukemia injection. Expression of Tgfb1 was quantified by qRT-PCR in MKs incubated with or without leukemia cells (three independent experiments). Significant differences: * p < 0.05, **p < 0.01, ***p <  Error bars represent SEM. AML = acute myeloid leukemia; BM = bone marrow; ELISA = enzyme-linked immunosorbent assay; FACS = fluorescence-activated cell sorting; MKs = megakaryocytes; MSCs = mesenchymal stem cells; qRT-PCR = quantitative real-time polymerase chain reaction; rhTPO = recombinant human thrombopoietin; rmSCF = recombinant mouse stem cell factor; TGFβ1 = transforming growth factor β1.
Experimental Hematology  , e2DOI: ( /j.exphem )
Copyright © 2018 ISEH – Society for Hematology and Stem Cells Terms and Conditions

5. Figure 3
Inhibitory effect of TGFβ1 on HSC proliferation via Egr3. (A) Left: Representative images of single-cell colony from CD34–LKS+ cells cultured for 10 days with or without TGFβ1. Right: Single-cell colony diameter was measured by AxioVision (two independent experiments). (B) Cell cycle analysis of LKS+ cells cultured for 48 hours with or without TGFβ1 (three independent experiments). (C) Expression of Egr3 and p21 was quantified by qRT-PCR in LKS+ cells incubated with or without TGFβ1 for 1 hour (three independent experiments). (D) Predicted SMAD2/3/4 complex binding site at −354 to −342 bp of mouse Egr3 gene according to the JASPAR database. (E) Enrichment of SMAD3 compared with IgG at the predicted binding site in (D), quantified by ChIP-PCR (three independent experiments). Significant differences: **p < 0.01, ***p <  Error bars represent SEM. ChIP = chromatin immunoprecipitation; HSCs = hematopoietic stem cells; ns = no significance; TGFβ1 = transforming growth factor β1.
Experimental Hematology  , e2DOI: ( /j.exphem )
Copyright © 2018 ISEH – Society for Hematology and Stem Cells Terms and Conditions

6. Supplementary Figure E1
FACS gating strategy for sorting of multiple types of niche cells.
Experimental Hematology  , e2DOI: ( /j.exphem )
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7. Supplementary Figure E2
Percentage (A) and total number (B) of megakaryocytes in AML (day 7) versus healthy control bone marrow.
Experimental Hematology  , e2DOI: ( /j.exphem )
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8. Supplementary Figure E3
Design of ChIP primer for detection of SMAD3 binding site in the promoter region of mouse Egr3 gene.
Experimental Hematology  , e2DOI: ( /j.exphem )
Copyright © 2018 ISEH – Society for Hematology and Stem Cells Terms and Conditions