Fig. 4

FoxC1 overexpression in ECs promotes self-renewal and survival of MSCs under hypoxia. A Representative growth state and morphology of the MSCs without (monoculture) or with coculture with the ECs alone, adFoxC1-treated, and siFoxC1-treated ECs in 108 h of culture under hypoxia conditions. Transfection of adFoxC1 improves self-renewal of MSCs, and FoxC1 deficiency impaired this effect. Scale bars = 200 µm. B MSCs were cultured for 108 h under hypoxic conditions, and the number of cumulative population doublings was determined. C For cell cycle distribution, MSCs without (monoculture) or with coculture with ECs treated with or without adFoxC1 or siFoxC1 for 108 h were collected and detected by flow cytometry, the percentage of of MSCs at S phase of the cell cycle was statistically analyzed as a histogram. D The percentage of cells in S phase was determined in MSCs without (monoculture) or with coculture with the ECs treated with or without adFoxC1 or siFoxC1. E MSCs after 108 h of hypoxic culture were stained with anti-Ki67 antibodies and then counterstained with DAPI, followed by confocal microscopy. Scale bars = 200 µm. F The proliferation of MSCs cocultured with adFoxC1- treated ECs was remarkably higher than the other MSCs, as assessed using a CCK8 assay. G MSCs after 108 h of hypoxic culture were evaluate by using the TUNEL assay. TUNEL-positive cells were obviously decreased in the MSCs cocultured with adFoxC1- treated ECs as compared with the other MSCs. Scale bars = 200 µm. H Statistical analysis of the mean percentage of annexin V-positive cells relative to the total number of MSCs. FoxC1 overexpression in ECs reduced the apoptosis of MSCs, and FoxC1 deficiency increased apoptosis. All data are the means ± SEM. One-way ANOVA analysis was used in D, and Welch ANOVA analyses were performed in F and H. p < 0.05: *versus monoculture, ^†versus ECs alone, and ^‡versus adFoxC1 + ECs (n = 10 in each group)