Journal: Proceedings of the National Academy of Sciences of the United States of America
Article Title: Prognostic and biological significance of the proangiogenic factor EGFL7 in acute myeloid leukemia
doi: 10.1073/pnas.1703142114
Figure Lengend Snippet: EGFL7 stimulates the proliferation of human and mouse AML cells. (A and B) BM cells from WT (A) and MllPTD/WTFlt3ITD/WT (B) mice were treated without (Unstim) or with 0.1, 0.25, or 0.5 μM rEgfl7 in Iscove’s Modified Dulbecco’s Medium (IMDM) + 2% BSA for 24, 48, 72, or 96 h. At the indicated time points, the number of viable cells was determined by Trypan blue dye exclusion assay. Each condition was repeated in triplicate. *P < 0.05, **P < 0.01; NS, not significant. (C) Blasts of the indicated AML patients (20,000 cells) were mixed with methylcellulose medium in the absence or presence of 0.25 μM rEGFL7 and were plated onto 2-cm dishes for 10 d. Colonies with more than 50 cells were enumerated using a light microscope. Each condition for each patient (n = 4) was plated in triplicate; **P < 0.01, ***P < 0.001. (D) Kasumi-1 cells were stimulated with 100 nM rEGFL7 for 4 h in RPMI1640 with 10% FBS. Cell proliferation was assessed using APC-BrdU/7AAD staining coupled with flow cytometry; *P < 0.05. (E) Kasumi-1 cells (2,500 cells) were mixed with methylcellulose medium in the absence or presence of 100 nM recombinant human EGFL7 and were scored after 10 d. Each condition was plated in triplicate in three independent experiments. *P < 0.05. (F) Blasts from AML patients (n = 4) were cultured in SFEM + 2% BSA in the absence or presence of 0.25 μM rEGFL7 for 20 min. Total proteins were extracted for immunoblotting of pAKT-S473 and total AKT. GAPDH was used as loading control. (G) AML blasts from MllPTD/WTFlt3ITD/WT mice (n = 3) were cultured in IMDM medium + 2% BSA in the absence or presence of 0.25 μM rEgfl7 for 20 min. Total proteins were extracted for immunoblotting of pAkt-S473 and total Akt. Gapdh was used as loading control. (H) Exponentially growing Kasumi-1 cells were starved in serum-free RPMI1640 medium for 1 h, followed by the addition of 100 nM recombinant EGFL7 for 5 min. Total proteins were extracted for immunoblotting of pAKT-S473, total AKT, and GAPDH.
Article Snippet: Egfl7 immunohistochemistry was performed with goat polyclonal anti-mouse Egfl7 (R-12) antibody (catalog no. sc-34416; Santa Cruz Biotechnology) at 1:50 dilution using the avidin–biotin complex method.
Techniques: Modification, Exclusion Assay, Light Microscopy, Staining, Flow Cytometry, Recombinant, Cell Culture, Western Blot, Control
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![EGFL7 inhibition results in decreased human AML cell growth without affecting normal hematopoietic cells. (A) Blasts from AML patients were cultured in SFEM with 10% FBS in the presence of 50 μg/mL of normal human IgG or anti-EGFL7 (@E7) antibody for 1 h. Total proteins were extracted for immunoblotting of pAKT-S473 and total AKT. GAPDH was used as loading control. (B) Human primary blasts (400,000) from AML patients (n = 4) were treated with 2, 10, 50, or 250 μg/mL of IgG control or anti-EGFL7 antibody in SFEM containing 10% FBS and cytokines for 2 h. Twenty thousand cells were plated in triplicate in methylcellulose medium and scored after 14 d of culture for mean ± SD colony numbers. *P < 0.05, **P < 0.01. (C) Forty-eight hours after IgG control vs. anti-EGFL7 antibody treatment (50 μg/mL) of AML cell lines, apoptosis was evaluated by Annexin V/7AAD staining, (D and E) Cell proliferation was measured using BrdU incorporation (D), and differentiation analysis was evaluated by CD11b expression (E). CD11b is depicted as the ratio of the CD11b expression value of the examined sample to the CD11b expression value of the corresponding IgG-treated control. *P < 0.05, **P < 0.01. (F) CD34+ CB cells from four different donors were plated in methylcellulose medium in the presence of increasing concentrations (2, 10, 50, or 250 μg/mL) of human IgG or anti-EGFL7 and were scored after 14 d of culture. Along with total number of colonies, colony types [erythroid burst-forming units (BFU), granulocyte/monocyte (GM), or granulocyte/erythrocyte/monocyte/megakaryocyte colonies (GEMM)] were enumerated also.](https://pub-med-central-images-cdn.bioz.com/pub_med_central_ids_ending_with_8639/pmc05468639/pmc05468639__pnas.1703142114fig05.jpg)