Fig. 6. Inhibition of internalize abolishes the tumour suppressor effects of SorCS3.

A, B Transwell assays were used to determine the influence of U87 and U251 cells pretreated with or without the cell-permeable dynamin inhibitor Dynasore (40 µM) for 2 h and then stimulated with NGF (100 ng/mL) for 60 min in overexpression of SorCS3 condition. C, D EdU staining to determine the influence of SorCS3 overexpression pretreated with or without the cell-permeable dynamin inhibitor Dynasore (40 µM) for 2 h and then stimulated with NGF (100 ng/mL) for 60 min, and EdU incorporation was calculated as EdU+ cells/total cells, quantified by ImageJ. Red was stained for proliferation (EdU + ), blue was stained for nucleus. E U87 cells were stimulated with Dynasore (40 µM) for 2 h and then stimulated with NGF (100 ng/mL) for 60 min, and then immunofluorescence for p75^NTR, early endosome markers (EEA1) and lysosome markers (Lamp1 and Lamp2). Scale bar: 17 µm. F–H U87 were pretreated with the cell-permeable dynamin inhibitor Dynasore (40 µM) for 2 h and then stimulated with NGF (100 ng/mL) for 60 min in overexpression of SorCS3 condition. Cell lysates were analyzed by western blotting with the indicated antibodies, using β-actin as an endogenous control. I, J U87 were pretreated with the Ro 08-2750 (1 µM, Ro 08-2750 is a reversible NGF inhibitor which inhibits NGF binding to p75^NTR selectively) for 8 h in overexpression of SorCS3 condition. Cell lysates were analyzed by western blotting with the indicated antibodies, using β-actin as an endogenous control. Data are shown as mean ± S.D. including three independent experiments. ns: not statistically significant.