Amotl1

AMOTL1 (HPA001196, Sigma), YAP1 (ab52771, Abcam), CTGF antibody (sc-14939, Santa Cruz), Ki67 (550609, BD Pharmingen), and cleaved-Caspase 3 (#9664, CST) were commercially available. The immunohistochemistry and the scoring of the results were performed as previously described [13 (link)]. For immunocytochemistry studies, AGS was cultured on coverslips in a six-well plate. After removal of the medium and three times of PBS washing, the cells were fixed with 4% paraformaldehyde at room temperature for 15 min. Followed by three times of PBS washing, the cells were then permeated with 0.1% Triton X-100 at room temperature for 15 min, and another PBS washing three times. Room-temperature blocking was done with 2% BSA for 45 min; then cells were incubated with the primary antibody (1:200) at 4 °C overnight. Again, after one time of PBS washing, the cells were incubated with goat anti-mouse IgG secondary antibody (Alexa Fluor 594, 1:400, Thermo Fisher Scientific) in the dark at room temperature for 1 h. After washing, nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI, Thermo Fisher Scientific). Images were captured with a microscope (Carl Zeiss Axio Imager 2, Oberkochen, Germany).

Cells were washed in PBS and lysed in M-PER^™ Mammalian Protein Extraction Reagent (ThermoFisher Scientific) containing the Halt^™ Protease Inhibitor Cocktail (ThermoFisher Scientific). Nuclear extraction was performed using NE-PER^™ Nuclear and Cytoplasmic Extraction Reagents (ThermoFisher Scientific). Protein concentration was quantified using the Pierce^™ Microplate BCA Protein Assay Kit (ThermoFisher Scientific). Membranes were blocked in in 5% non-fat milk for 1 hour and incubated with the following primary antibodies: YAP (1:1000; Cell Signaling Technology), YAP/TAZ (1:1000; Cell Signaling Technology), AMOTL1 (1:1000; Sigma-Aldrich, St. Louis, MO), AMOTL2 (1:1000, Sigma-Aldrich), p-YAP(Ser127) (1:1000; Cell Signaling Technology), Histone H3 (1:2000; Cell Signaling Technology), β-tubulin (1:1000; abcam), t-AKT (1:1000; Cell Signaling Technology), p-AKT (1:1000; Cell Signaling Technology), t-ERK (1:1000; Cell Signaling Technology), and p-ERK (1:1000; Cell Signaling Technology). β-actin (1:5000; Sigma-Aldrich, St. Louis, MO) and GAPDH (1:10000; EMD Millipore, Billerica, MA) were used as a loading controls. Each primary antibody was followed by incubation with secondary antibody (1:5000; Jackson ImmunoResearch Laboratories Inc. West Grove, PA) for 1 hour and bands were revealed with the Super Signal West Dura (ThermoFisher Scientific).