Fig. 3

Epidermal-growth factor receptor (EGFR) activation. A Screening of receptor tyrosine kinases (RTK) phosphorylation levels in DRG cultures exposed to osteoclast secretome. Images of the X-ray films. For the analysis, 100 µg of protein lysate from 3 independent experiments (n = 3), was pooled. Elliptical shapes highlighting the spots corresponding to epidermal-growth factor receptors (EGFR, ErbB2 and ErbB3, light green) and platelet-derived growth factor receptor-alpha (PDGFα, light purple). B Heatmap representing the relative spot intensity for the activated receptors calculated from the pixel density, showing the primary activation of two different families: EGFR family and PDGF. C Pharmacological inhibition of EGFR and ErbB2 with increasing doses of Erlotininb. Representative images of DRG treated with different concentrations of Erlotinib for 72 h (βIII tubulin in green and nuclei in blue, scale bar 500 µm). D Quantification of axonal outgrowth of sensory neurons blocked with EGFR inhibitor—Erlotinib at different concentrations added to osteoclast conditioned medium. Data represented as violon plot *p ≤ 0.05. E Levels of receptor tyrosine kinases (RTK) phosphorylation in DRG cultures exposed to osteoclast secretome (OC, blue) and EV-depleted secretome (EV-dep, light orange). Images of the X-ray films. Elliptical shapes highlighting the spots corresponding to epidermal-growth factor receptors (EGFR, ErbB2 and ErbB3, light green) and platelet-derived growth factor receptor-alpha (PDGFα, light purple). F Graph representing the mean spot intensity of the activated receptors EGFR, ErbB2 and PDGFα for the DRG exposed to osteoclast secretome (OC, blue) and EV-depleted secretome (EV-dep, light orange). Data represented as bars with individual values (n = 4), mean ± SD, ns—non-significative; ****p ≤ 0.0001. G Representative images of sensory neurons growth cones exposed to neurobasal (NB; upper row) vs. EV enriched fraction (EV+; lower row), stained against growth-associated protein (GAP-43, red) and phosphorylated PKCα (green); scale bar: 10 µm. H Quantification of the integrated intensity of phosphorylated PKCα at the growth cones exposed to NB (grey) vs. EV+ (orange). Intensity of phosphorylated PKCα normalized for the growth cone area calculated through GAP-43 staining. Results are presented as scatter dot plot; ***p ≤ 0.01