Fig. 4

ET-1 signaling blockage with macitentan decreases in vitro GBC cell invasion. a 1TKB (blue), 2TKB (red), NOZ (purple) and CAVE1 (yellow) cells were seeded in a matrigel-coated transwell chamber in FBS free medium, while the lower bottom contained medium + 10% FBS as chemoattractant. Cells were incubated at 37 °C for 16 h in presence or absence of 1 µM MAC. Cells were fixed and stained with crystal violet and counted using the 10× objective in 5 different fields. Control was normalized to 1 and then plotted as fold changes. Scale bar: 100 nm. Data represent averages ± SEMs (n = 3). ANOVA and Student’s tests were used. *p < 0.05 **p < 0.01. b MMP9 transcript levels were measured by RT-qPCR in two GBC cell lines, 1TKB (blue) and 2TKB (red), treated with 1 µM MAC for 24 h. Data represent averages ± SDs (n = 3)