Figure 3
Figure 3. Response of VWF expression to knockdown of miR-24 in mice. (A) Anti-miR-24 LNA (n = 8) or negative control (n = 8) injected into C57BL/6J mice. qPCR analysis of miR-24 expression in mice in heart, lung, brain, and kidney (normalized to U6); ****P < .0001; ***P < .001. (B) qPCR analysis of miR-24 expression in plasma of C57BL/6J mice treated with negative control (n = 8) or treated with anti-miR-24 LNA (n = 6) for 2 weeks (normalized to cel-miR-238 spike-in); **P < .01. (C) qPCR analysis of VWF mRNA levels in C57BL/6J mice heart, lung, brain, and kidney (n = 8 in each group); ****P < .0001; **P < .01; *P < .05. (D) ELISA analysis of VWF levels in plasma of mice with LNA and control treatment (n = 6-8 in each group); **P < .01. (E) Bleeding times of individual mice treated with negative control (n = 8) or anti-miR-24 LNA (n = 7) for 2 weeks; **P < .01. (F) Correlation between mice tail bleeding time and plasma miR-24 in C57BL/6J mice (anti-miR-24 LNA: n = 8; negative scrambled control: n = 5; and saline control: n = 4). (G) qPCR analysis of miR-24 expression in plasma of db/db mice treated with mimic control (n = 6) or treated with miR-24 mimic (n = 6) for 2 weeks (normalized to cel-miR-238 spike-in); **P < .01. (H) ELISA analysis of VWF levels in plasma of db/db mice treated with mimic control (n = 6) or treated with miR-24 mimic (n = 6) for 2 weeks (n = 6-8); *P < .05. (I) A photograph of a freshly dissected mouse aorta demonstrating sites of sections obtained for staining in J-L. (J) Mice were killed 2 weeks after LNA administration, and the arteries were thoroughly perfused, excised, fixed with 4% paraformaldehyde in PBS, embedded with optimum cutting temperature compound, snap-frozen, sectioned, and stained with anti-VWF fluorescein isothiocyanate (FITC) antibody; representing 2 thoracic aortas from negative control (n = 8) and 2 thoracic aortas from anti-miR-24 LNA (n = 8); scale bars, 50 µm. (K) Staining with anti-CD41 FITC antibody and anti-VWF phycoerythrin antibody; representing thoracic aorta from negative control (n = 8) and from anti-miR-24 LNA (n = 8). White dashed line indicates VWF and platelet thrombus; scale bars, 50 µm. (L) Staining with anti-CD41 FITC antibody and anti-VWF phycoerythrin antibody; representing aortic arch from negative control (n = 8) and from anti-miR-24 LNA (n = 8) (diagonal section shown to highlight thrombus). White dashed line indicates VWF and platelet thrombus; scale bars, 50 µm.

Response of VWF expression to knockdown of miR-24 in mice. (A) Anti-miR-24 LNA (n = 8) or negative control (n = 8) injected into C57BL/6J mice. qPCR analysis of miR-24 expression in mice in heart, lung, brain, and kidney (normalized to U6); ****P < .0001; ***P < .001. (B) qPCR analysis of miR-24 expression in plasma of C57BL/6J mice treated with negative control (n = 8) or treated with anti-miR-24 LNA (n = 6) for 2 weeks (normalized to cel-miR-238 spike-in); **P < .01. (C) qPCR analysis of VWF mRNA levels in C57BL/6J mice heart, lung, brain, and kidney (n = 8 in each group); ****P < .0001; **P < .01; *P < .05. (D) ELISA analysis of VWF levels in plasma of mice with LNA and control treatment (n = 6-8 in each group); **P < .01. (E) Bleeding times of individual mice treated with negative control (n = 8) or anti-miR-24 LNA (n = 7) for 2 weeks; **P < .01. (F) Correlation between mice tail bleeding time and plasma miR-24 in C57BL/6J mice (anti-miR-24 LNA: n = 8; negative scrambled control: n = 5; and saline control: n = 4). (G) qPCR analysis of miR-24 expression in plasma of db/db mice treated with mimic control (n = 6) or treated with miR-24 mimic (n = 6) for 2 weeks (normalized to cel-miR-238 spike-in); **P < .01. (H) ELISA analysis of VWF levels in plasma of db/db mice treated with mimic control (n = 6) or treated with miR-24 mimic (n = 6) for 2 weeks (n = 6-8); *P < .05. (I) A photograph of a freshly dissected mouse aorta demonstrating sites of sections obtained for staining in J-L. (J) Mice were killed 2 weeks after LNA administration, and the arteries were thoroughly perfused, excised, fixed with 4% paraformaldehyde in PBS, embedded with optimum cutting temperature compound, snap-frozen, sectioned, and stained with anti-VWF fluorescein isothiocyanate (FITC) antibody; representing 2 thoracic aortas from negative control (n = 8) and 2 thoracic aortas from anti-miR-24 LNA (n = 8); scale bars, 50 µm. (K) Staining with anti-CD41 FITC antibody and anti-VWF phycoerythrin antibody; representing thoracic aorta from negative control (n = 8) and from anti-miR-24 LNA (n = 8). White dashed line indicates VWF and platelet thrombus; scale bars, 50 µm. (L) Staining with anti-CD41 FITC antibody and anti-VWF phycoerythrin antibody; representing aortic arch from negative control (n = 8) and from anti-miR-24 LNA (n = 8) (diagonal section shown to highlight thrombus). White dashed line indicates VWF and platelet thrombus; scale bars, 50 µm.

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