Figure 4.
ITGB3+ MM cells adhere to immobilized EGFL7 via its RGD region (A) Fold change in ITGB3 expression of HUVEC, HEL, RPMI8226, U-266, MM.1S, and HS-5 cells when compared with ITGB3 expression in HL60 cells using RT-PCR. Transcripts normalized to b-ACTIN. Graphs represent averages from 3 independently prepared templates per condition. Experiments were repeated twice with similar results. (B) Representative FACS plots showing gating for PI negative cells. Histogram showing ITGB3 levels on indicated MM cell lines. (C) Percentage of adhesion of RPMI8226 cells after 4 hours on precoated plates with deposited ECM from cells infected with AdNull (Mock), AdEGFL7 full-form (EGFL7), or Addeleted RGD (delRGD), or precoated with rec. murine EGFL7 (rec. EGFL7) or with rec. FN. The percentage of adherent cells was quantified after cellular staining with crystal violet and absorbance measurement at 550 nm. Cumulative data of 2 independent experiments are shown (n = 5/condition per experiment). (D) Representative FACS histogram of ITGB3 expression using RPMI8226 cells stably transduced with ITGB3 (ITGB3 OE) in the left panel. The right panel shows the percentage of ITGB3 expression on ITGB3 OE cells, as determined by FACS (n = 6/group). (E) Representative western blot image of Tyr747, FAK EGFL7, and b-ACTIN proteins in EGFL7 OE/KD, and ITGB3 OE/KD in RPMI8226 cells. Two independent experiments were performed. (F) Human ITGB3 OE or ITGB3 KD cells were cultured on EGFL7- and FN-coated plates for 4 hours with/without the ITGB3 inhibitor Cilengitide (Celin). Quantification of adherence using crystal violet absorbance of adherent cells (n = 5 wells/condition). (G) Cell proliferation rate was determined after 24 hours by counting Trypan blue-negative ITGB3 OE and Mock MM cells (n = 3/wells per condition). (H) EGFL7 OE or EGFL7 KD MM cells were coinfected with Mock vector (−ITGB3) or ITGB3 OE (+ITGB3) vectors. Cells were counted after 24 hours in culture (n = 6/condition). Experiments were repeated at least twice. Graphs show mean ± SEM. Significance was calculated by Student t test, * P ≤ .05; ** P ≤ .01.

ITGB3+ MM cells adhere to immobilized EGFL7 via its RGD region (A) Fold change in ITGB3 expression of HUVEC, HEL, RPMI8226, U-266, MM.1S, and HS-5 cells when compared with ITGB3 expression in HL60 cells using RT-PCR. Transcripts normalized to b-ACTIN. Graphs represent averages from 3 independently prepared templates per condition. Experiments were repeated twice with similar results. (B) Representative FACS plots showing gating for PI negative cells. Histogram showing ITGB3 levels on indicated MM cell lines. (C) Percentage of adhesion of RPMI8226 cells after 4 hours on precoated plates with deposited ECM from cells infected with AdNull (Mock), AdEGFL7 full-form (EGFL7), or Addeleted RGD (delRGD), or precoated with rec. murine EGFL7 (rec. EGFL7) or with rec. FN. The percentage of adherent cells was quantified after cellular staining with crystal violet and absorbance measurement at 550 nm. Cumulative data of 2 independent experiments are shown (n = 5/condition per experiment). (D) Representative FACS histogram of ITGB3 expression using RPMI8226 cells stably transduced with ITGB3 (ITGB3 OE) in the left panel. The right panel shows the percentage of ITGB3 expression on ITGB3 OE cells, as determined by FACS (n = 6/group). (E) Representative western blot image of Tyr747, FAK EGFL7, and b-ACTIN proteins in EGFL7 OE/KD, and ITGB3 OE/KD in RPMI8226 cells. Two independent experiments were performed. (F) Human ITGB3 OE or ITGB3 KD cells were cultured on EGFL7- and FN-coated plates for 4 hours with/without the ITGB3 inhibitor Cilengitide (Celin). Quantification of adherence using crystal violet absorbance of adherent cells (n = 5 wells/condition). (G) Cell proliferation rate was determined after 24 hours by counting Trypan blue-negative ITGB3 OE and Mock MM cells (n = 3/wells per condition). (H) EGFL7 OE or EGFL7 KD MM cells were coinfected with Mock vector (−ITGB3) or ITGB3 OE (+ITGB3) vectors. Cells were counted after 24 hours in culture (n = 6/condition). Experiments were repeated at least twice. Graphs show mean ± SEM. Significance was calculated by Student t test, * P ≤ .05; ** P ≤ .01.

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