Figure 1.
EGFL7 is a survival factor in myeloma cells (A) Fold change in EGFL7 gene expression of the human stromal cell line HS-5 and the MM cell lines U-266, RPMI8226, (abbreviated RPMI), MM.1S, H929, and KMS11, as determined by RT-PCR when compared with the EGFL7 expression in the human myeloid leukemia HL60 cells. (B) A representative western blot is shown for EGFL7, with b-ACTIN as a control, in tumor lysates from mice injected with indicated RPMI cells. (C) Representative images of RPMI8226 cells stained for EGFL7 (green fluorescence) and 4′,6-diamidino-2-phenylindole (DAPI; blue nuclear staining) by immunofluorescent staining. (D) Fold change in EGFL7 gene expression of MACS-isolated CD138+ cells from normal donors when compared with the EGFL7 expression in CD138− cells. (E) Fold change in EGFL7 gene expression as determined by RT-PCR in human cell samples of patients with MM (patients #1 and #2 MM patient sample at diagnosis and patient #3 MM patient sample at refractory stage of the disease; for more clinical details, see supplemental Figure 1) when compared with EGFL7 gene expression found in human BMMCs of healthy donors (n = 3/condition). (F) Western blot analysis of EGFL7 and b-ACTIN as a control in indicated cell population from healthy volunteers and patients with MM. (Upper) Band quantification using the ImageJ program. (Lower) Representative western blots of the same samples. (G-H) EGFL7 (EGFL OE), EGFL7 knockdown (KD), or Mock MM cells (RPMI8226, MM.1S, and U-266) were generated. (G) Fold change in EGFL7 gene expression when compared with Mock cells, as determined by RT-PCR (n = 3/condition). (H) Cells were counted 24 hours after cell seeding after trypan blue exclusion (n = 6/group). (I-J) RPMI8226 cells (OE, KD, Mock) were stained with Annexin V-FITC and PI and analyzed 48 hours after cell plating by FACS. (I) A representative FACS blot is shown for each condition (n = 6/group). (J) Percentage of Annexin V+ and PI− cells (as a measure of early and late apoptosis) by FACS after 48 hours (n = 3/group). (K) Representative western blot analysis of the expression of phosphorylated AKT, BAX, and b-ACTIN (control) in cell lysates of Mock, EGFL7 OE, and EGFL7 KD RPMI8226 cells. (L) Fold change in caspase 3/7 activity of EGFL7 OE or KD cells when compared with Mock RPMI8226 cells 48 hours after cell plating, as determined by Versa Max (n = 6/condition). (M-N) EGFL7 OE and wild-type (RPMI) cells were treated with various concentrations of the AKT1/2 inhibitor. (M) Proliferation was determined using the CCK-8 kit after 24 hours in culture. % Absorbance indicates the rate of viable cells (n = 6/condition). (N) Fold increase in EGFL7 expression after AKT1/2 inhibitor treatment when compared with EGFL7 expression in nontreated cells. (O-P) MM cells were cocultured with a confluent layer of GFPpos-EGFL7 Mock, EGFL7 OE, or EGFL7 KD ECs in direct contact, and the percentage of GFP-MM cells was determined after 24 hours of coculture (n = 3/condition). (O) Percentage of GFP− MM cells in the adherent fraction. (P) Absolute number of nonadherent suspension cells retrieved from the cocultures. As for RT-PCR data, transcripts were normalized to b-ACTIN. Graphs represent averages from 3 independently prepared templates per condition. Experiments were repeated twice with similar results. Data are represented as mean ± SEM. * P ≤ .05; ** P ≤ .01; *** P ≤ .001. P values were determined using a Student t test.

EGFL7 is a survival factor in myeloma cells (A) Fold change in EGFL7 gene expression of the human stromal cell line HS-5 and the MM cell lines U-266, RPMI8226, (abbreviated RPMI), MM.1S, H929, and KMS11, as determined by RT-PCR when compared with the EGFL7 expression in the human myeloid leukemia HL60 cells. (B) A representative western blot is shown for EGFL7, with b-ACTIN as a control, in tumor lysates from mice injected with indicated RPMI cells. (C) Representative images of RPMI8226 cells stained for EGFL7 (green fluorescence) and 4′,6-diamidino-2-phenylindole (DAPI; blue nuclear staining) by immunofluorescent staining. (D) Fold change in EGFL7 gene expression of MACS-isolated CD138+ cells from normal donors when compared with the EGFL7 expression in CD138− cells. (E) Fold change in EGFL7 gene expression as determined by RT-PCR in human cell samples of patients with MM (patients #1 and #2 MM patient sample at diagnosis and patient #3 MM patient sample at refractory stage of the disease; for more clinical details, see supplemental Figure 1) when compared with EGFL7 gene expression found in human BMMCs of healthy donors (n = 3/condition). (F) Western blot analysis of EGFL7 and b-ACTIN as a control in indicated cell population from healthy volunteers and patients with MM. (Upper) Band quantification using the ImageJ program. (Lower) Representative western blots of the same samples. (G-H) EGFL7 (EGFL OE), EGFL7 knockdown (KD), or Mock MM cells (RPMI8226, MM.1S, and U-266) were generated. (G) Fold change in EGFL7 gene expression when compared with Mock cells, as determined by RT-PCR (n = 3/condition). (H) Cells were counted 24 hours after cell seeding after trypan blue exclusion (n = 6/group). (I-J) RPMI8226 cells (OE, KD, Mock) were stained with Annexin V-FITC and PI and analyzed 48 hours after cell plating by FACS. (I) A representative FACS blot is shown for each condition (n = 6/group). (J) Percentage of Annexin V+ and PI− cells (as a measure of early and late apoptosis) by FACS after 48 hours (n = 3/group). (K) Representative western blot analysis of the expression of phosphorylated AKT, BAX, and b-ACTIN (control) in cell lysates of Mock, EGFL7 OE, and EGFL7 KD RPMI8226 cells. (L) Fold change in caspase 3/7 activity of EGFL7 OE or KD cells when compared with Mock RPMI8226 cells 48 hours after cell plating, as determined by Versa Max (n = 6/condition). (M-N) EGFL7 OE and wild-type (RPMI) cells were treated with various concentrations of the AKT1/2 inhibitor. (M) Proliferation was determined using the CCK-8 kit after 24 hours in culture. % Absorbance indicates the rate of viable cells (n = 6/condition). (N) Fold increase in EGFL7 expression after AKT1/2 inhibitor treatment when compared with EGFL7 expression in nontreated cells. (O-P) MM cells were cocultured with a confluent layer of GFPpos-EGFL7 Mock, EGFL7 OE, or EGFL7 KD ECs in direct contact, and the percentage of GFP-MM cells was determined after 24 hours of coculture (n = 3/condition). (O) Percentage of GFP− MM cells in the adherent fraction. (P) Absolute number of nonadherent suspension cells retrieved from the cocultures. As for RT-PCR data, transcripts were normalized to b-ACTIN. Graphs represent averages from 3 independently prepared templates per condition. Experiments were repeated twice with similar results. Data are represented as mean ± SEM. * P ≤ .05; ** P ≤ .01; *** P ≤ .001. P values were determined using a Student t test.

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