Fig. 2

FoxC1 overexpression induces hypoxic EC proliferation. A Dynamic analysis of cell proliferation index assessed as the percentage of BrdU-positive cells to total number of cells was determined by FACS in the individual groups at 12-h, 24-h, 36-h, and 48-h after different treatments. B Equal protein loading was assessed by immunoblotting for FoxC1, and proliferation marker protein Ki67 at 48 h after treatment. Successful overexpression of the FoxC1 protein was verified by immunoblot analysis in the HO + adFoxC1 group. Note that FoxC1 overexpression strongly induced the expression of Ki67 in this group, while significant decreases in the levels of these proteins were observed in the HO + siFoxC1 group. C Fluorescence microscope images of the cells double stained with DAPI and Ki67, showing the greatest positive staining in the HO + adFoxC1 group, and the lowest in the HO + siFoxC1 group. Scale bars: 200 µm. D A percentage of Ki67 positively stained cells was expressed a proliferative index. E The proliferative capacities of ECs were also evaluated by CCK-8 assay in optical density (OD) value. Note that all these proliferative indexes of ECs showed the greatest levels in the HO + adFoxC1 group, the second highest in the NO + adFoxC1 group, and the smallest in the HO + siFoxC1 group. Data are shown as means ± SEM. Welch ANOVA analyses were performed in A and E, and one-way ANOVA analysis was used in D. p < 0.05: *versus at 12-h post-treatment, ^†versus 24-h post-treatment, ^‡versus 36-h post-treatment, ^§versus the normoxia, ^‖versus the NO/HO group under the same culture conditions, ^¶versus the siFoxC1 group (n = 10, each group)