Figure 5.
EGFL7 loss in MM cells overrides BTZ resistance (A-B) RPMI8226, U-266, and MM.1S cells were cultured for 24 hours in the presence of BTZ at indicated concentrations. (A) Fold change in EGFL7 gene expression of BTZ-treated MM cells when compared with non-BTZ-treated cultures, as determined by RT-PCR. Transcripts normalized to β-ACTIN. Graphs represent averages from independently prepared templates per condition. (B) Viable MM cells were counted 24 hours after BTZ treatment, using Trypan blue (n = 6/condition). (C) RPMI8226 cells were treated with rec. EGFL7 in the presence/absence of 5 nM BTZ. Cells were counted after 24 hours in culture (n = 6/group). (D) EGFL7 OE and Mock RPMI8226 cells were treated with/without BTZ. Cells were counted after 24 hours (n = 6/group). (E) RPMI Mock cells were cultured with/without neutralizing Abs against EGFL7 in the presence or absence of 10 nM BTZ. Cell proliferation was assessed using the CCK-8 kit after 24 hours in culture (n = 6/condition). (F-G) RPMI8226, U-266, and MM.1S cells were treated with/without BTZ for 24 hours. (F) Fold change in ITGB3 expression in BTZ-treated cells when compared with non-BTZ-treated cells after 24 hours determined by RT-PCR. Gene expression was normalized to β-ACTIN expression. Graphs represent averages from 3 independently prepared templates per condition. Experiments were repeated twice with similar results. (G) Representative FACS histograms showing ITGB3 expression in PI-negative gated cells after being cultured with or without BTZ for 24 hours. (H) Percentage of ITGB3-positive cells in BTZ or non-BTZ cultures, as determined by FACS (n = 6/condition). (I) Mock, EGFL7 KD, and EGFL7 OE RPMI cells were treated with/without BTZ for 24 hours in vitro. Washed RPMI8226 OE, Mock, or KD cells were allowed to adhere to plastic for 4 hours. Adherent cells were quantified using a fluorescence plate reader. Results are shown as percentage adhesion over the input. Experiment was run twice in triplicate (n = 6/condition). (J) Absolute number of GFP+ RPMI8226 cells per well after 4 hours of culture on EGFL7 OE or EGFL7 KD BMEC1 cells. (K) RPMI8226 cells were cultured in the presence or absence of BTZ with/without the integrin inhibitor Cilengitide (Celin; n = 6/group). The number of viable cells was counted after 24 hours. The experiment was run once in triplicate (n = 6/condition). (L) Mock, ITGB3 OE, and ITGB3 KD RPMI cells were treated with/without BTZ. Viable cells were counted using the Trypan exclusion assay (n = 6/group). The experiment was repeated twice. (M) Mock, EGFL7 OE, and EGFL7 KD RPMI cells concomitantly infected with ITGB3 OE or KD plasmids were treated for 24 hours with/without BTZ (n = 6/group). The number of viable cells was determined. The experiment was performed twice. (N) Study treatment scheme. Mock, EGFL7 OE, and EGFL7 KD RPMI MM cells concomitantly infected with/without ITGB3 OE or KD plasmids were injected s.c. into NOD/SCID mice, and tumors were established. Starting on the 25th day (treatment day 1), tumor-carrying mice were randomized according to tumor size. A total of 5 injections of PBS/dimethyl sulfoxide carrier, or BTZ (1 mg/kg) were given twice per week (n = 5 mice per group). Tumor weight was determined on day 36 (n = 5-7/group). Graphs show mean ± SEM. Significance was calculated by Student t test, * P ≤ .05; ** P ≤ .01.

EGFL7 loss in MM cells overrides BTZ resistance (A-B) RPMI8226, U-266, and MM.1S cells were cultured for 24 hours in the presence of BTZ at indicated concentrations. (A) Fold change in EGFL7 gene expression of BTZ-treated MM cells when compared with non-BTZ-treated cultures, as determined by RT-PCR. Transcripts normalized to β-ACTIN. Graphs represent averages from independently prepared templates per condition. (B) Viable MM cells were counted 24 hours after BTZ treatment, using Trypan blue (n = 6/condition). (C) RPMI8226 cells were treated with rec. EGFL7 in the presence/absence of 5 nM BTZ. Cells were counted after 24 hours in culture (n = 6/group). (D) EGFL7 OE and Mock RPMI8226 cells were treated with/without BTZ. Cells were counted after 24 hours (n = 6/group). (E) RPMI Mock cells were cultured with/without neutralizing Abs against EGFL7 in the presence or absence of 10 nM BTZ. Cell proliferation was assessed using the CCK-8 kit after 24 hours in culture (n = 6/condition). (F-G) RPMI8226, U-266, and MM.1S cells were treated with/without BTZ for 24 hours. (F) Fold change in ITGB3 expression in BTZ-treated cells when compared with non-BTZ-treated cells after 24 hours determined by RT-PCR. Gene expression was normalized to β-ACTIN expression. Graphs represent averages from 3 independently prepared templates per condition. Experiments were repeated twice with similar results. (G) Representative FACS histograms showing ITGB3 expression in PI-negative gated cells after being cultured with or without BTZ for 24 hours. (H) Percentage of ITGB3-positive cells in BTZ or non-BTZ cultures, as determined by FACS (n = 6/condition). (I) Mock, EGFL7 KD, and EGFL7 OE RPMI cells were treated with/without BTZ for 24 hours in vitro. Washed RPMI8226 OE, Mock, or KD cells were allowed to adhere to plastic for 4 hours. Adherent cells were quantified using a fluorescence plate reader. Results are shown as percentage adhesion over the input. Experiment was run twice in triplicate (n = 6/condition). (J) Absolute number of GFP+ RPMI8226 cells per well after 4 hours of culture on EGFL7 OE or EGFL7 KD BMEC1 cells. (K) RPMI8226 cells were cultured in the presence or absence of BTZ with/without the integrin inhibitor Cilengitide (Celin; n = 6/group). The number of viable cells was counted after 24 hours. The experiment was run once in triplicate (n = 6/condition). (L) Mock, ITGB3 OE, and ITGB3 KD RPMI cells were treated with/without BTZ. Viable cells were counted using the Trypan exclusion assay (n = 6/group). The experiment was repeated twice. (M) Mock, EGFL7 OE, and EGFL7 KD RPMI cells concomitantly infected with ITGB3 OE or KD plasmids were treated for 24 hours with/without BTZ (n = 6/group). The number of viable cells was determined. The experiment was performed twice. (N) Study treatment scheme. Mock, EGFL7 OE, and EGFL7 KD RPMI MM cells concomitantly infected with/without ITGB3 OE or KD plasmids were injected s.c. into NOD/SCID mice, and tumors were established. Starting on the 25th day (treatment day 1), tumor-carrying mice were randomized according to tumor size. A total of 5 injections of PBS/dimethyl sulfoxide carrier, or BTZ (1 mg/kg) were given twice per week (n = 5 mice per group). Tumor weight was determined on day 36 (n = 5-7/group). Graphs show mean ± SEM. Significance was calculated by Student t test, * P ≤ .05; ** P ≤ .01.

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