Fig. 3

YAP1 of Hippo signaling is the direct target of miR-141 (A) The schematic drawing showed YAP1 constructs with wild-type (YAP1-WT) and mutant (YAP1-MT) miR-141 binding sites paired with the miR141 sequence (upper). Dual-luciferase assay showed the relative luciferase activities that cotransfection of pmir-YAP1-WT or pmir-YAP1-MT and the miR-141 expressing plasmid pmR-141 (pmR-ZsGreen1 empty vector was used as negative control) in WPMY-1 and HEK293 cell lines (mean ± SEM, t-test, *P < 0.05, **P < 0.01). N = 3 independent experiments. (B) Confirmation of miR-141 as a target of YAP1 by dose-dependent transfection of pmR-Zsgreen1-miR141 (0, 0.5, 1.0, and 2.0 μg) into WPMY-1, T HESC and WI-38 cells by western blot analysis. The relative YAP1 expression (YAP1/β-actin) was quantified by ImageJ software. (C) Graphic charts compared the relative transcription level of GROα between WPMY-1 stromal cells with either shRNA-mediated knockdown of YAP1 (YAP1-KD), CRISPR/Cas9 system-mediated knockout of YAP1 (YAP1^low/−) or knockout of TAZ (TAZ^low/− #1 and TAZ^low/− #2) with the respective control (Ctrl) (upper). QPCR and ELISA analyses showed the relative expression of GROα in WPMY-1 YAP1^low/− cells transfected with the YAP1-expressing plasmid (0, 1 and 2 μg) (lower) (mean ± SEM, t-test, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001). N = 3 independent experiments. (D) Western blot analysis showed the changes in YAP1/TAZ in the cytoplasm and nucleus in WPMY-1 cells with shRNA-mediated YAP1 knockdown approach (YAP1-KD). β-actin and Lamin A/C were used as the internal controls for the cytosol and nuclear proteins, respectively. (E) Immunofluorescence microscopy showed changes in YAP1/TAZ in the cytoplasm and nucleus in YAP1 knockdown and miR-141-overexpressing WPMY-1 cells, and parental cells were used as a control. Scale bar = 20 μm. (F) The graph showed the relative percent input of ChIP that indicated the interaction between TEAD and the predicted binding sites on GROα in T HESCs cells with TEAD overexpression and vector control (mean ± SEM, t-test, *P < 0.05). (G) Graphic charts show the relative luciferase activity of cotransfected TEAD1, TAZ and pGL3-GROα (N ~ III) in the WPMY-1 cells by dual-luciferase assay (mean ± SEM, one-way ANOVA, *P < 0.05, ***p < 0.001, ****p < 0.0001). N = 3 independent experiments