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: YAP1 transcriptional regulation of AGK expression in gastric cancer cells. A, The levels of AGK and CTGF proteins were analysed by using Western blot in YAP-siRNA or cDNA-transfected HGC-27 and BGC-823 cells. B, HGC-27, BGC-823 and MKN-45 cells were treated with metformin (0, 10, 20 or 50 mmol L-1) and verteporfin (0, 5, 10 or 20 µmol L-1) for 48 h and analysed for YAP1, AGK and CTGF expression. C, qRT-PCR results indicated that after knocking down YAP1, AGK along with Hippo-YAP1 target gene mRNAs, such as for CTGF and CYR61, were decreased in HGC-27 cells, while overexpressing YAP1 increased AGK, CTGF and CYR61 mRNA levels in BGC-823 cells. D, AGK promoter activity was detected in HEK-293T cells that transfected with YAP1 cDNA, YAP-S94A or the combination of YAP1 and TEAD1-4 cDNA. E, Illustration of luciferase reporter constructs (wild-type and mutated-type). Mutant luciferase constructs were generated by site-directed mutagenesis of TRE1 or TRE2 alone or in combination. F, HEK293T cells were grown and cotransfected with the indicated plasmids and then subjected to luciferase reporter assay. The mutated TRE1 in the AGK promoter was sufficient to abolish AGK promoter activity induced by YAP/TEAD4 cDNA. G, Design of the AGK promoter primers for ChIP assays. H, Chromatin from HGC-27 was pulled down using a YAP1 antibody or IgG. PCR amplification using TRE1 or TRE2 primers was then performed. A 292 bp PCR product containing TRE1 in the AGK promoter was amplified from the chromatin DNA that was pulled down by the YAP antibody, while the input chromatin is shown as a positive control for ChIP assay

Fig 4: AGK protein up-regulation in gastric cancer tissues and the association with clinicopathological parameters from patients. A, Analysis of the TCGA data. The alteration frequency of AGK in various cancer tissues. The data were modified from the cBioPortal for Cancer Genomics (http://www.cbioportal.org/). B, Analysis of the TCGA data. Expression of AGK mRNA was significantly higher in gastric cancer tissues (n = 285) than that in normal tissues (n = 33). C and D, Western blot and immunohistochemistry results of AGK protein levels in GC tissues and adjacent non-cancerous gastric tissues. AGK protein levels are higher in GC tissues (T) than in adjacent normal gastric tissues (N) from the same patient (n = 12). E and F, An immortal gastric epithelial cell line (GES-1) and five gastric cancer cell lines (MKN-45, HGC-27, SGC-7901, BGC-823 and MGC-803) were grown and subjected to protein and mRNA extraction for AGK and YAP1. G, Immunohistochemical staining of AGK in the formalin-fixed, paraffin-embedded gastric cancer tissues and normal gastric tissues. H, Kaplan-Meier curves stratified by AGK expression in 120 cases of gastric cancer patients. I, Kaplan-Meier curve stratified by AGK expression in gastric cancer patients obtained from the TCGA-STAD database

Fig 5: 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