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. 2017 Jul 14;24(10):1811–1820. doi: 10.1038/cdd.2017.121

Figure 3.

Figure 3

ET-1R/β-arr1 axis induces β-catenin pathway in CRC-SC. (a) β-catenin localization evaluated by immunofluorescence staining (green) in CC09 cells stimulated for 30 min with ET-1 (100 nM) alone or in combination with MAC (1 μM). Nuclei were counterstained with DAPI (blue) (scale bar, 10 μm). (b) IB analysis for β-catenin and β-arr1 protein expression in cytoplasmic and nuclear extract of CC09 cells treated with ET-1(100 nM) for the indicated times. HSP-70 and PCNA were used as cytoplasmic and nuclear loading control, respectively. (c) IB analysis for β-catenin and β-arr1 protein expression in nuclear extracts of CC09 cells transfected with SCR or si-β-arr1 and treated for 30 min with ET-1 (100 nM) and/or MAC (1 μM). Histone H3 (H3) was used as loading control. (d) Nuclear extracts of CC09 cells, treated for 30 min with ET-1 (100 nM) and/or MAC (1 μM), were immunoprecipitated (IP) with anti-β-arr1 or with irrelevant IgG (IgG) and immunoblotted with anti-β-catenin and anti-β-arr1. H3 was used as loading control. (e) β-catenin transcriptional activity evaluated in CC09 cells transfected with SCR or si-β-arr1 and treated for 24 h with ET-1 (100 nM) and/or MAC (1 μM). Values are shown as mean±S.D. from three independent experiments repeated in triplicates (*P<0.001 versus CTR; **P<0.002 versus ET-1 in SCR-transfected cells). (f) ET-1 promoter activity evaluated in CC09 cells treated as in (e). 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 in SCR-transfected cells). ChIP analysis performed in CC09 cells treated for 30 min with ET-1 (100 nM) and/or MAC (1 μM) (g) or in CC09 cells transfected with SCR or si-β-arr1 and treated for 30 min with ET-1 (100 nM) and/or MAC (1 μM) (h). Chromatin was incubated with β-arr1, β-catenin, p300, H3K27ac and TCF4 Abs and analyzed by PCR analysis by using specific primers for ET-1 promoter. Non-specific immunoglobulin G (IgG) was used as irrelevant Ab (IRR). The input DNA lane represents one-twentieth of the precleared chromatin used in each ChIP reaction. (i) ET-1, Cyclin D1 and Axin 2 mRNA expression in CC09 cells stimulated for 24 h with ET-1 (100 nM) alone or in combination with MAC (1 μM) evaluated by qPCR, normalized using endogenous cyclophilin-A. Values are shown as mean±S.D. from three independent experiments repeated in triplicates (*P<0.01 versus CTR; **P<0.05 versus ET-1)