Figure 2.

CM collected from the GATA6-overexpressing clones increase both the mRNA levels and activities of MMP-9 in HUVECs and the latter is critical for their tube formation. (A) Total RNAs (5 µg) isolated from HUVECs after being cultured in the CM collected respectively from the 116 vec, OE4, and OE6 as well as the 29 vec, OEC, and OED clones for 48 h were subjected to RT-qPCR analyses to determine the mRNA levels of MMP-2 and -9 genes. Data are the mean  ±  SD of three independent experiments. *** p < 0.005 compared with the respective vector clones by Student’s t-test. N.S. indicates no significant difference compared also with the respective vector clones. (B) The activities of MMP-2 and MMP-9 released from HUVECs after being cultured in the CM collected respectively from the HCT-116 (left) as well as the HT-29 (right) for 24 h were examined by gelatin zymography as described in the “Materials and methods”. The quantitative results shown in the bar graphs (lower panels) are the mean  ±  SD of three independent experiments. * p < 0.05, ** p < 0.01, and *** p < 0.005 compared with the respective vector clones by Student’s t-test. (C) Tube formation assays of HUVECs were carried out primarily as described above using the CM collected from three clones derived respectively from HCT-116 and HT-29 cells without or with MMP9 inhibitor II (1 µM). The quantitative results shown in the lower panels are the mean  ±  SD of three independent experiments. * p < 0.05, ** p < 0.01, and *** p < 0.005 compared with the respective untreated vector clones by Student’s t-test. ### p < 0.005 compared with the corresponding untreated groups by Student’s t-test.