Figure 1.

Expression of ET-1/β-arr1 axis in CRC-SC. (a) In a panel of patient-derived CRC-SC (CC09, CSC5, CSC2 and CSC1), expression of ET-1, ET_AR, ET_BR and β-arr1 mRNA copy number was analyzed by real-time PCR (qPCR) normalized using endogenous cyclophilin-A. Values are shown as mean±S.D. from three independent experiments repeated in triplicates. (b) ET_AR, ET_BR and β-arr1 protein expression was analyzed by immunoblotting (IB). Tubulin was used as loading control. (c) ET-1 secretion evaluated by ELISA in 24 h cell-conditioned media. Values are shown as mean±S.D. from three independent experiments repeated in triplicates. (d) Time-dependent effect of treatment with ET-1 (100 nM) and/or macitentan (MAC) (1 μM) on cell growth of CC09 cells transfected with siRNA negative control (SCR) or with β-arr1 siRNA (si-β-arr1). Values are shown as mean±S.D. from three independent experiments repeated in triplicates (*P<0.001 versus CTR; **P<0.001 versus ET-1) (e) Sphere formation assay of CSC5 cells, treated with ET-1 (100 nM) and MAC (1 μM) alone or in combination for 7 days. Values are shown as mean±S.D. from three independent experiments repeated in triplicates (*P<0.05 versus CTR; **P<0.05 versus ET-1). Representative images of tumor spheres were shown in the right panel