Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Dsg2 via Src-mediated transactivation shapes EGFR signaling towards cell adhesion

doi: 10.1007/s00018-018-2869-x

Figure Lengend Snippet: Dsg2 knockout results in mislocalization and reduced protein levels of EGFR in intestinal epithelial cells. a Immunostaining for Dsg2 (green) and EGFR (red) in patient tissue sections of the colon revealed co-localization of both proteins. Four independent patient tissue samples were analyzed. Shown is representative image of a section from the colon. Bar 10 µm. b Immunostaining for Dsg2 (green) and EGFR (red) in human enteroids revealed co-localization of both proteins along the cell borders as well as on the surface facing the lumen. Shown is representative image for ten enteroids. Bar 10 µm. c Confluent cell monolayer of DLD1 cells grown on coverslips were immunostained for EGFR, Dsg2, and Dsc2/3. Loss of Dsg2 but not Dsc2/3 results in a major loss of EGFR staining at cell borders. Bar 10 µm. d Merged images of immunofluorescence staining of Dsg2, EGFR, and Dsc2/3 show co-localization of Dsg2 and EGFR but not Dsc2/3 and EGFR. Bar 10 µm (left panel) and 5 µm (right panel). e Evaluation of the Pearson correlation coefficient of EGFR confirms a co-localization of EGFR with Dsg2 but not Dsc2. Shown are boxplots with each point representing one analyzed area along the cell border. *p < 0.05. f STED super resolution microscopy analysis of Dsg2 and EGFR co-immunostaining shows co-localization. Bar 5 µm. g x–z image of Dsg2 and EGFR co-immunostaining shows specific co-localization at the apical site of cell contacts. Bar 10 µm (left panel) and 5 µm (right panel). h EGFR is absent in the Triton X-100 insoluble fraction when Dsg2 is missing in contrast to Ecad that is not affected by Dsg2 knockout. GAPDH served as loading control. i Band intensity of detected EGFR was measured from five independent experiments, showing a significant reduction of EGFR in the insoluble fraction in Dsg2-deficient DLD1 cells. Results are shown as mean ± SE. *p < 0.05. j Total protein level of EGFR in DLD1 cells were assessed by western blotting that revealed reduced EGFR levels in Dsg2-deficient cells. GAPDH served as loading control. k Band intensity of detected EGFR was quantified from at least ten independent experiments, demonstrating a significant reduction of total EGFR protein levels upon Dsg2 knockout. Shown are mean ± SE. *p < 0.05

Article Snippet: For western blot analysis and immunofluorescence staining, following primary antibodies were used: mouse anti-Dsg2 (clone 10G11) and rabbit anti-Dsg2 (rb5, both Progen, Heidelberg, Germany), mouse anti-Dsc2/3 (clone 7G6) and rabbit anti Claudin-4 (both Life Technologies, Carlsbad, CA, USA), rabbit anti-DP I/II (H-300), rabbit anti-EGFR (clone 1005-G), mouse anti-EGFR (clone A-10) and mouse anti-GAPDH (all from Santa Cruz Biotechnology, Santa Cruz, CA, USA), mouse anti-α-tubulin (Abcam, Cambridge, UK), and rabbit anti-phospho-EGFR Tyr845 and rabbit anti-Src (both from Cell Signaling, Danvers, MA, USA).

Techniques: Knock-Out, Immunostaining, Staining, Immunofluorescence, Microscopy, Western Blot

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Dsg2 via Src-mediated transactivation shapes EGFR signaling towards cell adhesion

doi: 10.1007/s00018-018-2869-x

Figure Lengend Snippet: EGF reduces Dsg2-specific binding events on the cell surface of living DLD1 cells. a Dsg2 adhesion measurements were performed on living DLD1 cells under control conditions and after incubation with EGF or erlotinib. Shown are topography images of selected areas at cell borders and adhesion maps with each white pixel representing one binding event. b Quantification shows a significant reduction of binding events on living DLD1 cells after incubation with both, EGF or erlotinib. Application of EGF also reduced the amount of binding events in the cell-free AFM setup with a Dsg2-coated tip and EGFR-coated mica sheets. Graph bars represent fold-change values ± SE from at least four independent experiments. *p < 0.05. c Peak fit analysis of measured unbinding forces revealed a distribution peak of 31.56 pN under control conditions. d Distribution-peak values of unbinding forces measured on DLD1 cells under control conditions and after incubation with EGF, anti-EGFR antibody, or erlotinib were compared and revealed similar values for all conditions. Graph shows distribution-peak values normalized to control. e, f Hanging drop bead aggregation assay confirms blockade of interaction between Dsg2 and EGFR through EGF. Beads coated with a human IgG Fc fragment served as control. *p < 0.05; n.s. not significant. g Western blot analysis of surface biotinylation assay revealed reduced Dsg2 and EGFR levels on the surface of DLD1 cells after incubation with EGF. h Quantification of Dsg2 and EGFR band intensity from at least seven independent experiments shows a significant reduction of surface protein levels only after incubation with EGF. Graph bars represent fold-change values ± SE. *p < 0.05

Article Snippet: For western blot analysis and immunofluorescence staining, following primary antibodies were used: mouse anti-Dsg2 (clone 10G11) and rabbit anti-Dsg2 (rb5, both Progen, Heidelberg, Germany), mouse anti-Dsc2/3 (clone 7G6) and rabbit anti Claudin-4 (both Life Technologies, Carlsbad, CA, USA), rabbit anti-DP I/II (H-300), rabbit anti-EGFR (clone 1005-G), mouse anti-EGFR (clone A-10) and mouse anti-GAPDH (all from Santa Cruz Biotechnology, Santa Cruz, CA, USA), mouse anti-α-tubulin (Abcam, Cambridge, UK), and rabbit anti-phospho-EGFR Tyr845 and rabbit anti-Src (both from Cell Signaling, Danvers, MA, USA).

Techniques: Binding Assay, Incubation, Western Blot, Surface Biotinylation Assay