Figure 3. Src mediates knockdown of DDR1-induced E-cadherin endocytosis.

(a) Mock and Sh-DDR1 cells were seeded on a culture dish at the indicated points of time, and levels of Src-pY418, c-Src, DDR1, and E-cadherin in whole cell lysate were analysed using Western blotting. The quantified result of Src-pY418/c-Src is shown in (b). Each bar represents mean ± SE of three independent experiments. *Indicates P < 0.05, **indicates P < 0.01 to its paired control. (c) The localisations of Src-pY418 in Mock and Sh-DDR1 cells were assessed using immunofluorescence staining after cells were cultured for 24 h. The arrow heads mark the junctional colocalisation of E-cadherin and Src-pY418 in the XY section, and the arrows mark the colocalisation of E-cadherin and Src-pY418 in the XZ section. The dashed lines in the XY section indicate the position of the XZ section. Bar: 10 μm. (d) Mock and Sh-DDR1 cells were cultured for 24 h and then treated with 10 μM PP3 (control) or PP2 for 4 h. Cells were fixed and immunostained for E-cadherin (green), α-catenin (red), and nuclei (blue). (e) The fluorescence intensity profiles of the colocalisation of E-cadherin and β-catenin were assessed using Olympus FV-1000 imaging software. (f) The fluorescence intensity profiles of the Pearson’s correlation coefficient of E-cadherin and β-catenin were assessed using Olympus FV-1000 imaging software. **Represents P < 0.01; ***represents P < 0.001. (g) Mock and Sh-DDR1 cells were transiently transfected with HECD-mEosFP and incubated on chamber slides for 24 h. After PP3 (10 μΜ) or PP2 (10 μΜ) treatment for 4 h, Mock and Sh-DDR1 cells were subjected to photoconversion analysis and live images were captured using an Olympus FV-100 confocal microscope. Representative fluorescence images of three experiments during the recording time are shown. (h) The quantification results of (g) are shown as mean ± SE from three independent experiments in parallel with the treatment group.