American Society of Hematology

Figure 2

$Figure 2. NO regulates MT1-MMP activity and membrane clustering in migrating ECs. (A) Migrating HUVECs were treated with 100 μM DEA-NONOate (DEA) or DETA-NONOate (DETA) for 3, 6, or 16 hours, and membrane expression of MT1-MMP was detected by flow cytometry. The chart shows the mean plus or minus SD of the fold induction of the percent of MT1-MMP–positive cells from 3 independent experiments (left). None indicates nonstimulated controls. Total cellular expression of MT1-MMP was analyzed by Western blot in migratory HUVECs treated for 16 hours with 100 μM DETA-NONOate (DETA) or 20 ng/mL PMA (right). VE-cadherin is shown as loading control. The fold induction relative to nonstimulated (NS) cells was estimated from densitometric analysis of 5 independent experiments. (B) NO increases collagenolytic activity at the cell membrane of migratory ECs. HUVECs were seeded at subconfluence onto 10 μg/mL COL I or DQ-COL I and left untreated (None) or treated with 100 μM DETA-NONOate or 1 μM bradykinin for 3 or 6 hours. DQ-COL I cleavage was visualized by its fluorescent product. Data are the means plus or minus SD of the percent of cells showing DQ-COL I fluorescence (an average of 40 cells were counted per condition); *P < .05; **P < .02 (left). DQ-COL I cleavage at cell protrusions was increased in bradykinin-treated ECs (right; arrowheads). (C) NO increases membrane clustering of MT1-MMP. The subcellular location of MT1-MMP was detected by immunostaining of subconfluent HUVECs treated with 100 μM DEA-NONOate (DEA) and DETA-NONOate (DETA) for different periods (left). Two blinded observers independently quantitated the number of clusters per cell. Data show the means plus or minus SD of at least 80 cells per condition from 6 independent experiments. *P < .04; **P ≤ .02 (right). (D) The distribution of MT1-MMP between caveolar membrane and cytosolic fractions was assayed by cell membrane isolation with Triton X-114 from HUVECs treated with DEA-NONOate or DETA-NONOate as in panel C. One representative experiment of 4 performed is shown. The fold induction relative to nonstimulated (NS) cells was estimated from densitometric analysis.$

NO regulates MT1-MMP activity and membrane clustering in migrating ECs. (A) Migrating HUVECs were treated with 100 μM DEA-NONOate (DEA) or DETA-NONOate (DETA) for 3, 6, or 16 hours, and membrane expression of MT1-MMP was detected by flow cytometry. The chart shows the mean plus or minus SD of the fold induction of the percent of MT1-MMP–positive cells from 3 independent experiments (left). None indicates nonstimulated controls. Total cellular expression of MT1-MMP was analyzed by Western blot in migratory HUVECs treated for 16 hours with 100 μM DETA-NONOate (DETA) or 20 ng/mL PMA (right). VE-cadherin is shown as loading control. The fold induction relative to nonstimulated (NS) cells was estimated from densitometric analysis of 5 independent experiments. (B) NO increases collagenolytic activity at the cell membrane of migratory ECs. HUVECs were seeded at subconfluence onto 10 μg/mL COL I or DQ-COL I and left untreated (None) or treated with 100 μM DETA-NONOate or 1 μM bradykinin for 3 or 6 hours. DQ-COL I cleavage was visualized by its fluorescent product. Data are the means plus or minus SD of the percent of cells showing DQ-COL I fluorescence (an average of 40 cells were counted per condition); *P < .05; **P < .02 (left). DQ-COL I cleavage at cell protrusions was increased in bradykinin-treated ECs (right; arrowheads). (C) NO increases membrane clustering of MT1-MMP. The subcellular location of MT1-MMP was detected by immunostaining of subconfluent HUVECs treated with 100 μM DEA-NONOate (DEA) and DETA-NONOate (DETA) for different periods (left). Two blinded observers independently quantitated the number of clusters per cell. Data show the means plus or minus SD of at least 80 cells per condition from 6 independent experiments. *P < .04; **P ≤ .02 (right). (D) The distribution of MT1-MMP between caveolar membrane and cytosolic fractions was assayed by cell membrane isolation with Triton X-114 from HUVECs treated with DEA-NONOate or DETA-NONOate as in panel C. One representative experiment of 4 performed is shown. The fold induction relative to nonstimulated (NS) cells was estimated from densitometric analysis.

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