Extended Data Fig. 4: EGF-induced de-sialylation triggers retrograde trafficking to the Golgi.

a, Retrograde trafficking of Gal3. HeLa cells stably expressing GalT–GFP-SNAP were continuously incubated for 4 h at 37 °C with benzylguanine (BG)-labelled Gal3 in the presence or absence of 100 ng/ml EGF. GFP-trap pulldown samples were analysed by western blotting with the indicated antibodies. Note the reduced electrophoretic mobility of the conjugate between Gal3 and GalT–GFP-SNAP (red dashed box). Representative blot of one out of 3 independent experiments. b, Retrograde trafficking analysis by immunofluorescence. HeLa cells stably expressing GalT–GFP-SNAP were continuously incubated for 1 h at 37 °C with benzylguanine (BG) and Seta555-labelled Gal3. Cells were fixed and analysed by confocal microscopy. Note that the fraction of internalized Gal3 in perinuclear Golgi increases upon incubation with 100 ng/ml of EGF. Representative maximum intensity images of 3 independent experiments are shown. Unpaired t-test, minimum of 50 cells per condition. c, Cluster analysis of the summarized list of biological process gene ontology phrases associated with the list of 26 Gal3 binders that were significantly enriched after EGF stimulation. Spot size equals -LOG2 P value, spot colour equals -LOG10 GO annotations. d, Retrograde trafficking of α3 integrin. HeLa cells stably expressing GalT–GFP-SNAP were continuously incubated for 4 h at 37 °C with benzylguanine (BG)-labelled anti-α3 integrin antibody (ASC-1) in the presence or absence of 100 ng/ml EGF. The anti-α3 integrin antibody–SNAP conjugate in GFP-trap pulldowns on lysates from the indicated conditions of 4 independent experiments was quantified by western blotting. Note that EGF increases the retrograde trafficking of α3 integrin. Unpaired t-test. e–h, Cell migration on cell-driven matrices. MDA-MB-231 cells were seeded in 1% FCS on cell derived matrices and cell migration was monitored for 16 h at 37 °C in the presence or absence of indicated inhibitors. Velocity and accumulated distance were calculated from 3 independent experiments (≈50 cells analysed per condition), using the manual tracking software ImageJ. One-way ANOVA with Dunnett’s multiple comparison test. For all quantifications in this figure, means ± SEM are shown. In (e–h), means from separate experiments are indicated by solid dots, and measurements of individual cells have different coloured symbols for each experiment. ****p < 0.0001. Source numerical data and unprocessed blots are provided.

Source data