Fig 2: FYN directly binds TOPK and phosphorylates TOPK at Y272. A Ni–NTA-His-TOPK bound with endogenous FYN of MGC803 cells. B COIP experiments confirm that FYN can bind to TOPK endogenously. C Colocalization of FYN and TOPK was visualized by confocal microscope in MGC803 cells. Cytoplasmic and nuclear staining of FYN and TOPK was mostly merged together. D Active FYN phosphorylated inactive TOPK in vitro in the presence of [?-32P] ATP as visualized by autoradiograph. E Potential phosphorylated tyrosine sites of TOPK were predicted by NetPhos 3.1 software program. F FYN phosphorylated TOPK at Y272 in peptide mapping. Five synthesized peptides containing potential tyrosine sites were used as substrates in an in vitro kinase assay with active FYN in the presence of [?-32P] ATP and the results were visualized by autoradiography. G Wild type His-TOPK (WT), single mutant His-TOPK (74F), single mutant His-TOPK (272F) or double mutant His-TOPK (FF) were used as substrates in an in vitro kinase assay with active FYN in the presence of [?-32P] ATP and the results were visualized by autoradiography. H Validation of anti phospho-TOPK (Y272) (p-TOPK (Y272)) in an in vitro kinase assay. Wild type His-TOPK (WT), single mutant His-TOPK (74F), single mutant His-TOPK (272F) or double mutant His-TOPK (FF) as shown was used as substrate for active FYN. Reactive products were resolved by SDS-PAGE and visualized by Western blot with p-TOPK (Y272). I Co-transfection of Flag-FYN and HA-TOPK-WT,HA-TOPK-Y272F in 293 T cells. J-K Detection of changes in TOPK, p-TOPK(Y272) levels after knockdown of FYN expression in SGC7901 and MGC803 cells

Fig 3: FYN is highly expressed in GC and is strongly associated with poor prognosis in GC patients. A The FYN genetic alterations (gene amplification, deep deletion, or somatic mutation) and mRNA expression in GC samples from the TCGA cohort (total alteration rate: 7%). B FYN mRNA expression is upregulated in the tumor tissues compared with it in normal tissue group from TCGA. C FYN mRNA high expression GC has poor overall survival. D FYN mRNA high expression GC has poor progression-free survival. E–F FYN expression was higher in gastric cancer tissues than in paraneoplastic tissues (p < 0.05). G Overexpressed FYN was associated with poor overall survival in GCs (p < 0.05).H. Specificity of ROC curve to detect FYN as a diagnostic marker for GC

Fig 4: HSPB1 acts as a downstream effector of FYN/TOPK and mediates its biological function. A Colocalization of FYN and HSPB1 was visualized by confocal microscope in MGC803 and SGC7901 cells. Cytoplasmic and nuclear staining of FYN and TOPK was mostly merged together. B Colocalization of TOPK and HSPB1 was visualized by confocal microscope in AGS cells. C-D CCK-8 assay detected cell viability in Control group, shFYN group, shFYN + OE-TOPK group and shFYN + OE-TOPK + HSPB1 inhibitor J2 group. 5 × 103 cells per well, N = 3, The data are expressed as the means ± standard deviations. *p < 0.05, **p < 0.01, ***p < 0.001, ns not significant. E Representative images of migration and invasion of Control group, shFYN group, shFYN + OE-TOPK group and shFYN + OE-TOPK + HSPB1 inhibitor J2 group in MGC803 cells. F Statistical results of the number of migrated cells in each group of MGC803 cells. The number of invaded cells per well was quantified. scale bar, 100 μm. N = 5, The data are expressed as the means ± standard deviations. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ns not significant. G Statistical results of the number of invasive cells in each group of MGC803 cells. The number of invaded cells per well was quantified. scale bar, 100 μm. N = 5, The data are expressed as the means ± standard deviations. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001,ns not significant. H Representative images of migration and invasion of Control group, shFYN group, shFYN + OE-TOPK group and shFYN + OE-TOPK + HSPB1 inhibitor J2 group in SGC7901 cells. I Statistical results of the number of migrated cells in each group of SGC7901 cells. The number of invaded cells per well was quantified. scale bar, 100 μm. N = 5, The data are expressed as the means ± standard deviations. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ns not significant. J. Statistical results of the number of invasive cells in each group of SGC7901 cells. The number of invaded cells per well was quantified. scale bar, 100 μm. N = 5, The data are expressed as the means ± standard deviations. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ns not significant

Fig 5: Knockdown of FYN inhibits the migration and invasion of gastric cancer cells. A Knockdown of FYN inhibited the migration and invasion ability of MGC803 cells. The number of invaded cells per well was quantified. scale bar, 100 μm. N = 5, The data are expressed as the means ± standard deviations. *p < 0.05, **p < 0.01, ***p < 0.001, ns not significant. B Knockdown of FYN inhibited the migration and invasion ability of SGC7901 cells. The number of invaded cells per well was quantified. scale bar, 100 μm. N = 5, The data are expressed as the means ± standard deviations. *p < 0.05, **p < 0.01, ***p < 0.001, ns not significant. C Scratch assay confirmed that knockdown of FYN significantly inhibited the migration ability of MGC803 and SGC7901 cells. The scratch area of 0H, 24H, 48H is calculated and quantified. N = 3, The data are expressed as the means ± standard deviations. *p < 0.05, **p < 0.01, ***p < 0.001, ns not significant