Fig 2: A proposed model depicts how VPS35 participates in the trafficking of EGFR and its effects on downstream ERK and AKT signalling.

Fig 3: VPS35 expression upregulated in tumour tissues and associated with poor prognosis of the patients with gastric cancer. (a) Upregulation of VPS35 in six digestive system cancer types in the GEPIA database. (b) Overall survival curves of the 384 patients in the gastric cancer cohort in GEPIA, with patients stratified based on VPS35 expression levels (Log-rank test). (c) Average immunohistochemistry (IHC) scores of VPS35 of patient samples in the Ruijin gastric cancer cohort (n = 96, Chi-square test). (d) Representative images of VPS35 IHC in gastric cancer and matched normal tissues from the Ruijin cohort. (e) Overall survival curves of our patient cohort, with patients stratified based on VPS35 expression levels (Log-rank test). (Scale bars: 100 μm, ∗, p < 0.05; ∗∗, p < 0.01; ∗∗∗, p < 0.001 and ∗∗∗∗, p < 0.0001.).

Fig 4: VPS35 promoted gastric cancer development in vivo. (a) Images of subcutaneous tumours formed by MKN28/Ctrl, MKN28/shVPS35, HGC27/Ctrl, and HGC27/VPS35 cells (five biological replicates). (b) Quantification (tumour volume at indicated time points, tumour weight at endpoint) of subcutaneous tumours formed by MKN28/Ctrl, MKN28/shVPS35, HGC27/Ctrl, and HGC27/VPS35 cells. (c) Representative images of H&E staining and IHC (VPS35 and p-CDK2) of subcutaneous tumours formed by MKN28/Ctrl, MKN28/shVPS35, HGC27/Ctrl, and HGC27/VPS35 cells. (Scale bars: 100 μm. The student's t tests, ∗, p < 0.05; ∗∗, p < 0.01; ∗∗∗, p < 0.001 and ∗∗∗∗, p < 0.0001.).

Fig 5: VPS35 recycled EGFR to the cell surface and decreased EGFR degradation. (a) VPS35 knockdown enhanced EGF-induced EGFR degradation, whereas VPS35 overexpression inhibited EGFR degradation. Western blotting detected the alterations in the total EGFR degradation in indicated cells in response to EGF stimulation in the presence of cycloheximide (left panels). Densitometric analysis of EGFR blots from three independent experiments are shown as mean ± SD (right panels). Experiments were conducted three times independently. (b) Localisation analysis of EGFR after EGF and cycloheximide treatment at different time points. At 0 min, EGFR was located mainly on the cell surface in all indicated cells after overnight starvation. With the passing of time, AGS/shVPS35 cells showed weaker fluorescence intensity compared to AGS/Ctrl cells, while HS-746T/VPS35 cells retained stronger fluorescence intensity than cells in the control group did. RFP: EGFR. Experiments were conducted three times independently. (c) VPS35 knockdown inhibited EGFR recycling and VPS35 overexpression promoted EGFR recycling. Indicated cell surfaces were labelled with Sulfo–NHS–SS-biotin on ice and stimulated for 30 min with EGF at 37 °C. Recycling of EGFR was determined at different time points indicated in the graphs by western blotting (upper panels). The values in western blotting were mean ± SD from three independent experiments (lower panels). Experiments were conducted three times independently. (Scale bars: 100 μm).