Fig. 2

Dual ETRs antagonism with macitentan (MAC) promotes ET-1 signaling downregulation in GBC cells. a Transcript levels of ET-1 target genes were measured by RT-qPCR in two GBC cell lines, 1TKB (blue) and 2TKB (red) treated with 1 µM MAC for 24 h. b Total β-catenin and NF-kB (p65) levels were measured by western blot in NOZ GBC cells treated with 100 nM ET-1 and/or 1 µM MAC for 24 h. Representative images from three independent experiments are shown. c Cytosolic and nuclear β-catenin and NF-kB (p65) were measured by western blot in 1TKB and 2TKB GBC cells treated with 1 µM MAC for 24 h. Lamin B1 was used as a nuclear marker. d Indirect immunofluorescence of NOZ cells treated with 1 µM MAC and/or 100 nM ET1 for 24 h. β-catenin was visualized using a specific antibody and the nucleus was stained with DAPI. e Cycloheximide stability assay of β-catenin in NOZ cells treated with 100 nM ET-1 and/or 1 µM MAC between 0 and 4 h. f Reporter activity of β-catenin was measured by luciferase reporter assay using the TOP/FOB Flash system. Values reported for luciferase activity were used to calculate the TOP/FOP ratios (mean ± SD). Representative images from three independent experiments are shown. Data represent averages ± SDs (n = 3). ANOVA and Student’s tests were used. *p < 0.05