Extended Data Fig. 1: EGF induces Gal3 binding to cells in vitro and in vivo.

a, Serum-starved MDA-MB-231 cells were incubated for 1 h on ice with 200 nM Alexa488-labelled Gal3 in the presence of EGF at the indicated concentrations. Fluorescence intensity was determined for 4 independent experiments (≈100 cells per condition). One-way ANOVA with Dunnett’s multiple comparison test. b, Gal3 (green) binding to HN12 cells. HN12 cells were incubated for 30 min at 37 °C with 200 nM AFT or 1 mM DANA in serum-free media before incubation for 1 h on ice in serum-free media with 200 nM Alexa488-labelled Gal3 in the presence or absence of 100 ng/ml EGF. Fluorescence intensity was determined from 6 independent experiments (≈200 cells per condition) and represented as %CTRL. One-way ANOVA with Dunnett’s multiple comparison test. SUM intensity confocal images are shown. Scale bar, 10 µm. Nuclei in blue. c, Gal3 binding to mouse embryonic fibroblasts (MEFs). MEFs were serum starved for 30 min at 37 °C in the presence or absence of sialidase (1 unit) or 1 mM DANA. In the continued presence or absence of sialidase or DANA, the cells were then incubated for 30 min on ice with 200 nM Alexa488-labelled Gal3 in the presence or absence of 100 ng/ml EGF. Total fluorescence/area from SUM confocal images was quantified from one representative experiment (≈30 cells per condition) out of two and represented as %CTRL. One-way ANOVA with Turkey’s multiple comparison test. Maximum intensity images are shown. Scale bar, 10 µm. d, Gal3 binding to NR6 cells. NR6 cells (mutant EGFR) and NR6 expressing functional EGFR were serum starved for 30 min at 37 °C before incubation for 30 min on ice with 200 nM Alexa488-labelled Gal3 in the presence or absence of 100 ng/ml EGF. Total fluorescence/area from SUM confocal images was quantified from one representative experiment (≈25 cells per condition) out of two and represented as %CTRL. One-way ANOVA with Turkey’s multiple comparison test. e, In vivo experiment in mice. 6 h after injection of 30 mg/kg AFT into mice, tongues were excised and lysed in RIPA buffer, lysates from each of the mice were probed with antibodies against pEGFR (Y1068) and tubulin. Data were normalized to first lane (cohort 1 orange triangle, cohort 2 blue triangles). Mann–Whitney test. f, EGF-induced signalling. MDA-MB-231 cells were serum starved with the indicated inhibitors for 1 h at 37 °C before incubation for 10 min at 37 °C in the continued presence or absence of the inhibitors and of 100 ng/ml EGF, as indicated. Cells were lysed in RIPA buffer, and lysates probed with the indicated antibodies. g,h, Quantification of 3 independent experiments as in (f). Determination of pERK signal (g) or the pRSK signal (h). One-way ANOVA with Tukey’s multiple comparison test. i, General inhibition of sialylation. MDA-MB-231 cells were incubated for 72 h at 37 °C with 10 µM sialostatin (STI) or DMSO, serum starved for 30 min at 37 °C and incubated for 1 h on ice with 200 nM Alexa488-labelled Gal3 in the presence or absence of 100 ng/ml EGF. Total fluorescence/area from SUM confocal images was quantified from 3 independent experiments (≈75 cells per condition) and represented as %CTRL. One-way ANOVA with Holm-Šídák’s multiple comparisons test. j, EGF effect on SNA binding. MDA-MB-231 cells were serum starved for 1 h at 37 °C before incubation for 1 h on ice with 5 µg/ml of SNA-Cy5 in the presence or absence of 100 ng/ml EGF. Total fluorescence/area from SUM confocal images was quantified from 3 independent experiments (≈75 cells per condition) and represented as %CTRL. One-way ANOVA with Dunnett’s multiple comparison test. k, EGF effect on Lectenz binding. MDA-MB-231 cells were serum starved for 1 h at 37 °C, incubated for 1 h on ice with 5 µg/ml of the α2-3 Sia linkage-specific biotinylated Lectenz reagent in the presence or absence of 100 ng/ml EGF, fixed and labelled with neutravidin-488. Total fluorescence/area from SUM confocal images was quantified from one representative experiment (≈50 cells per condition) out of 2 and represented as %CTRL. Unpaired t-test. l, EGF effect on RCA lectin binding. MDA-MB-231 cells were serum starved for 30 min in the presence of DANA before incubation on ice with 5 µg/ml biotin coupled RCA lectin in the presence or absence of EGF (100 ng/ml) and/or DANA. Total fluorescence/area from SUM confocal images was quantified from 4 independent experiments (≈100 per condition). One-way ANOVA, Dunnett’s multiple comparison test. For all quantifications except (e), means ± SEM are shown. For (e) the solid line indicates median of all data points. In (a,b,i–l), means from separate experiments are indicated by solid dots, and measurements of individual cells have different coloured symbols for each experiment. ns = p > 0.05, * p ≤ 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. Source numerical data and unprocessed blots are provided.

Source data