Phospho-MOB1A (Thr35) Antibody - #AF4481

FIGURE 5 EGCG promoted retention of YAP1 in the cytoplasm by activating the Hippo pathway and promoting autophagy in breast cancer cells. (A) Natural small‐molecule compounds predicted to activate the Hippo pathway in breast cancer cell lines. (B) Bioinformatics analysis of pathways related to EGCG. (C) WB analysis was performed to examine expression of Hippo pathway components and YAP1 target genes (CTGF and CYR61) in MCF7 and MDA‐MB‐231 cells treated with EGCG of various concentrations for 6 h. (D) Expression levels of YAP1 and p‐YAP1 were determined in the nucleus and cytoplasm of MCF7 and MDA‐MB‐231 cells via WB analysis. LaminB1 and β‐actin were used as extraction controls for the nucleus and cytoplasm, respectively. (E‐F) Expression levels of the autophagy markers LC3 and p62 were detected by WB in MCF7 and MDA‐MB‐231 cells after treatment with EGCG of various concentrations or for different lengths of time. (G) IF staining images showing LC3 fluorescence puncta and quantitative analysis of MCF7 and MDA‐MB‐231 cells treated with EGCG (50 µg/mL, 6 h). DAPI labelled with blue fluorescent signal was used to mark the nucleus, while green fluorescent signal was used to label LC3. (H) Autophagic structures (indicated with red arrows) were detected with TEM in MCF7 and MDA‐MB‐231 cells treated with EGCG (50 µg/ml, 6 h). Data are from three independent experiments and are shown as the mean ± standard deviation. **P < 0.01 (Student's t‐test). Abbreviations: YAP1, Yes1‐associated transcriptional regulator; EGCG, epigallocatechin gallate; WB, Western blotting; MST1, macrophage stimulating 1; p‐MST1, phosphorylated‐macrophage stimulating 1; MOB1A: MOB kinase activator 1A; p‐MOB1A: phosphorylated‐MOB1A; CTGF, connective tissue growth factor; CYR61, cysteine rich angiogenic inducer 61; LC3, microtubule‐associated protein 1 light chain 3; SQSTM1/p62, sequestosome 1; IF, immunofluorescence staining; DAPI, 4',6‐diamidino‐2‐phenylindole; TEM, transmission electron microscopy.