Fig 2: Association of YAP1 with AGK proteins in gastric cancer cells and tissues. A, The expression of YAP1 with AGK protein in gastric cancer tissues is shown in two representative cases. B, The correlation of staining intensity between YAP1 and AGK in gastric cancer tissues. C, Association of YAP1 with AGK expression in gastric cancer tissues from the TCGA-STAD database data, plotted in the GEPIA website. D, The protein expressions of AGK, YAP1 and CTGF were evaluated in HGC-27 cells that were transfected with non-target siRNA + pcDNA3.1 vector, AGK siRNAs + pcDNA3.1 vector or the combination of AGK siRNAs and YAP cDNA

Fig 3: AGK promotion of gastric cancer cell proliferation in vitro and in vivo. A, Cells were transfected with AGK siRNAs, cDNA or the negative control, and then subjected to Western blot. The efficiency of AGK knockdown and overexpression in HGC-27, BGC-823 and MKN-45 cells was determined. GAPDH was used as a loading control. B, Cells were transfected with AGK siRNAs, cDNA or the negative control, and then subjected to CCK-8 assay. Overexpression of AGK in BGC-823 and MKN-45 cells promoted cell proliferation rate, whereas knockdown of AGK in HGC-27 and MKN-45 cell inhibited cell proliferation rate, less significantly in YAP1-deficient (MKN-45) cells. C, Cells were transfected with AGK siRNAs, cDNA or the negative control, and then subjected to colony formation assay. Knockdown of AGK inhibits clonogenic ability of gastric cancer cells, overexpression of AGK promotes clonogenic ability of gastric cancer cells, less significantly in YAP1-deficient (MKN-45) cells. D-G, Nude mouse tumour cell xenograft assay. D, The images of xenograft tumours that were harvest at the end of experiments. E, Growth curves of xenograft tumours derived from HGC-27 cells that expressed LV-scramble-shRNA or LV-shAGK. F, Comparison of the average weight of collected tumours from above two groups. G, The protein levels of AGK, YAP1 and CTGF were detected in six tumour samples by WB analysis with indicated antibodies. The data are summarized as the mean ± SD of three independent experiments. *P < .05, **P < .01 by two-tailed t test

Fig 4: AGK inhibition of the Hippo-YAP1 pathway and up-regulation of TEAD transcriptional activity. A, B, Western blot analysis of MST1/2, LATS1/2, p-YAP, YAP1 and CTGF expression after transfection with AGK siRNAs (A) and plasmids carrying AGK cDNA (B) in HGC-27 and BGC-823 cells, respectively. C, Confocal images of YAP1 staining in BGC-823 cells after transfection with AGK siRNA or AGK cDNA. D, Relative TEAD luciferase reporter activity in AGK after transfection with AGK siRNA or negative control siRNA. E, AGK overexpression resulted in an increase in the expression of Hippo-YAP/TEAD target genes, including CTGF, CYR61, survivin and CDX2. Conversely, knockdown of AGK had the opposite effect on expression of these genes. F, Illustration of the YAP1-AGK positive feedback loop in gastric cancer cells