Figure 1.
Effect of MZ on MOVAS cell proliferation.
A. MOVAS cell proliferation by XTT. MOVAS cells were seeded in 96-well plate and incubated with 1 µM MZ for 48 hours. Cell proliferation was measured with an XTT proliferation kit and expressed as percentage of control group treated with DMSO. B. MOVAS cell proliferation by direct counting. MOVAS cells were seeded in 96-well plate and incubated with 1 µM MZ for 48 hours. MOVAS cells were detached and counted manually. Cell proliferation was expressed as percentage of control group treated with DMSO. C. Dose effect of MZ on MOVAS cell proliferation. MOVAS cells were seeded in 96-well plate and incubated with MZ at indicated concentration for 48 hours. Cell proliferation was measured with an XTT proliferation kit and expressed as percentage of control group treated with DMSO. D. Tubulin staining. MOVAS cells undergoing proliferation were immmunostained for tubulin (red) and nuclei (blue). Arrows point to cells undergoing different stage of cell division. Scale bar: 50 µm. **P<0.01.
Figure 2.
Effect of MZ on MOVAS cell migration.
A. MOVAS cell migration. Scratch injury was performed on a confluent cell monolayer. Cells were imaged at 0 and 24 hours after injury. B. Quantification of cell migration in wound healing assays after MZ treatment for 24 hours. Data are presented as percentages of the recovered scratch area relative to untreated control cells. C. Dose effect of MZ in wound healing assay. Effect of different MZ doses on cell migration. D. Transwell migration assay. The upper panels are representative images. The lower panel is the quantification result. E. Tubulin staining. MOVAS cells undergoing migration were immmunostained for tubulin (red) and nuclei (blue). Arrows point to polarized microtubules. F. Filamentous actin (F-actin) staining. MOVAS cells undergoing migration were immmunostained for filamentous actin (green) and nuclei (blue). Scale bar: 50 µm. **P<0.01.
Figure 3.
Effect of MZ on VSMC Apoptosis.
A. Dead cell staining. Dead cells were stained with trypan blue 24 hours after injury. B. Apoptosis cell staining. Cells undergoing apoptosis were stained 24 hours after injury. Arrow points to active caspase 3/7 positive cell. C. TUNEL staining. Arrow points to apoptotic cells 24 hours after injury. D. Dose effect of MZ on apoptosis at indicated concentration by TUNEL staining. Data are presented as percentage of total cells. E. Quantification of apoptotic cells treated with MZ or apoptosis inhibitor (Inh) in wound healing assay by TUNEL staining. Data are presented as percentages of the total cells. F. Quantification of cell migration in wound healing assay after MZ and apoptosis inhibitor (Inh) treatment for 24 hours. Data are presented as percentages of the recovered scratch area relative to untreated control cells. G. Quantification of cell proliferation after MZ and apoptosis inhibitor (Inh) treatment for 48 hours. Cell proliferation was measured with an XTT proliferation kit and expressed as percentage of control group treated with DMSO. H. Migrated cells treated with MZ. 24 hours after the injury in migration assay (left), cells were treated with 1 µM MZ for another 24 hours (right). Scale bar: 50 µm. *P<0.05, **P<0.01.
Figure 4.
Effect of MZ on intimal hyperplasia following femoral artery wire injury.
A. Sections stained for elastin. The area of intima and media was analyzed with ImagJ software. Arrows point to internal elastic lamina. B. VSMC α-actin immunostaining. Vascular smooth muscle cells (brown) were detected with α- SMC actin antibody. C. CD 31 immunostaining. Endothelial cells (brown) were detected with CD31 antibody. Arrows point to endothelium. D. PCNA immunostaining. Proliferating cells (brown) were detected with PCNA antibody. E. TUNEL staining. Apoptotic cells (green) were detected with TUNEL assay. Scale bar: 50 µm.
Table 1.
Statistical results of femoral artery wire injury assay.