Fig. 6: The integrin-FAK-SRC axis mediates VPS35-induced YAP activation in tumour cells.

A Venn diagram showing the proteins lost from the surface of VPS35-depleted cells. The expression of ITGB3, p-FAK, FAK, p-SRC, SRC, p-AKT and AKT was assessed by Western blotting in VPS35-overexpressing GC cells (B) and VPS35-knockdown GC cells (C). D Endogenous VPS35 was immunoprecipitated with an anti-ITGB3 antibody, with IgG as the negative control, and immunocomplexes were analysed via WB with anti-ITGB3 and anti-VPS35 antibodies. E Mass spectrogram of VPS35 phosphorylation. F Binding motif between VPS35 and ITGB3. G Endogenous ITGB3 was immunoprecipitated with an anti-VPS35 antibody with or without phosphorylase inhibitor, with cell lysate supernatant as the positive control, and immunocomplexes were analysed via WB with anti-ITGB3, anti-phosphoserine/threonine and anti-VPS35 antibodies. H VPS35-overexpressing or VPS35-knockdown cells and their corresponding control cells were surface-labelled with cleavable biotin at 4 °C, left alone to allow internalisation or recycling, and examined by Western blotting. I Quantification of recycled ITGB3 in VPS35-overexpressing or VPS35-knockdown cells and the corresponding control cells. J VPS35-overexpressing GC cells were treated with RGD as indicated, and the levels of p-FAK, FAK, p-AKT, AKT, p-LATS1, LATS1, p-MST1/2, MST1, p-YAP (ser127A) and YAP were determined by Western blotting. Immunofluorescence assay evaluating the nuclear translocation of YAP in VPS35-overexpressing NCI-NN87 cells with or without RGD treatment (K). Quantification of the ratio and the intensity of nuclear YAP staining (L). M–P Western blot analysis was performed to evaluate the correlations among ITGB3, VPS35, and YAP in GC tissues (n = 27). *p < 0.05, **p < 0.01.