Fig 1: CXCR7/ß-arr1-mediated biased signal induces YAP1 nuclear translocation in CRC cells. A Western blot analysis of YAP1 expression in cytoplasmic and nuclear extracts of HCT116Control and HCT116LV-CXCR7 cells treated with or without CXCL12 (100 ng/ml) for 60 min. GAPDH and Lamin B1 were used as cytoplasmic and nuclear loading control, respectively. B, C YAP1 localization evaluated by immunofluorescence (IF) in HCT116 and HT29 cells treated with or without CXCL12 (100 ng/ml). YAP1 was labeled with Alexa Fluor® 488 donkey anti-rabbit secondary antibodies, nuclei were visualized with DAPI, shown in blue. Scale bars, 50 µm. D Analysis of endogenous YAP1-ß-arr1 interaction in HCT116 cells by Co-immunoprecipitation (Co-IP). Normal rabbit IgG antibodies were used as control. E IF staining was performed to determine the colocalization of YAP1 (red) and ß-arrestin1 (green) or ß-arrestin2 (green) in HCT116 cells treated with CXCL12 (100 ng/ml) in the presence of AMD3100 (2 µM). DAPI was used for nuclear staining. Scale bars, 50 µm. F Western blot analysis of YAP1 and ß-arrestin1 expression in cytoplasmic and nuclear extracts of HCT116 and HT29 cells treated with or without CXCL12 (100 ng/ml) in the presence of AMD3100 (2 µM). GAPDH and Histone H3 were used as cytoplasmic and nuclear loading control, respectively. G Western blot analysis of ß-arrestin1 expression in HCT116 cells transfected with ß-arrestin1 siRNA. ß-actin was used as loading control. H Western blot analysis of YAP1 expression in cytoplasmic and nuclear extracts of HCT116 cells transfected with ß-arrestin1 siRNA or siNC and treated with or without CXCL12 (100 ng/ml) plus AMD3100 (2 µM). GAPDH and Histone H3 were used as cytoplasmic and nuclear loading control, respectively. Bars are means ± SD; *P < 0.05, **P < 0.01, ***P < 0.001, NS stands for no significance (n = 3)
Fig 2: PLEKHH2 binds to ß-arrestin1 through its FERM domain and promotes FAK phosphorylation, PI3K/AKT pathway activity, and cell malignant phenotype.A The GEPIA database showed that PLEKHH2 was significantly positively correlated with ARRB1 (ß-arrestin1) level. B Endogenous ß-arrestin1 and PLEKHH2 are co-localized in the cytoplasm. C Endogenous ß-arrestin1 interact with both PLEKHH2 and FAK in A549 and H1299 cells. D In lung cancer cells, transfection with the full-length PLEKHH2 decreased the interaction of ß-arrestin1 and FAK compared with that of the empty vector. Transfection with a PLEKHH2 mutant with a FERM domain deletion has no effect on decreasing ß-arrestin1 binding to FAK. E Western blotting showed that PLEKHH2-?FERM did not significantly enhance p-FAK, p-PI3K, p-AKT, and proliferation- and invasion-related proteins compared to transfection with full-length PLEKHH2. At the same time, transfection with PLEKHH2-?FERM had did not significantly promote cell proliferation, migration, and invasion, as evidenced by the results of the clone formation assay (F), CCK-8 assay (G), and Matrigel Transwell assay (H). P < 0.05 indicates statistical significance, *P < 0.05. Statistical analysis is shown in Supplementary Fig. 6 and Supplementary Fig. 7A-B.
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