Fig. 6

The impact of IVC on VM formation. a VM formation of MHCC97H cells cultured in a Matrigel-coated 24-well plate with culture medium containing IVC (7.5, 15 or 30 μM) or Y27632 (50 μm) for 8 h or 24 h. Photographs were taken at the indicated time points. Original magnification was 100×, scale bars represent 125 μm. b Quantitative analysis of the mean number of tube-like structures formed from six randomly chosen areas in 3D cultures. c Real-time qPCR analysis was used to determine changes in gene expression. Relative IVC-induced change in gene expression compared with GAPDH is expressed as fold change calculated by 2^-ΔΔCp method. Y27632 (50 μM) was used as positive control. d Representative confocal images (n = 3, five pictures per condition) of control and IVC (7.5, 15 or 30 μM) with 24 h incubation after immunostaining for VE-cadherin, PI3K, MMP-2 or MMP-9 (green) and DAPI (blue). Scale bars represent 50 μm. e Western blot assay was performed to evaluate the effect of IVC or Y27632 on VM markers, using GAPDH as an internal control for protein loading. f Relative protein level was quantitated using Image J. Data are expressed as mean ± S.E. from three independent experiments, with significant differences from control designated as *P < 0.05, **P < 0.01 and ***P < 0.001