Fig. 4

Expression of CD146 on cultured HDMECs and sorted BECs and LECs. A Flow cytometry analysis of freshly isolated HDMECs from human foreskin dermis. The hierarchical gating strategy involved the consecutive exclusion of debris, doublets and dead cells using Zombie Aqua staining. CD31 and Podoplanin were used to discriminate between BECs (CD31⁺Podoplanin⁻) and LECs (CD31⁺Podoplanin⁺). CD146 expression was detected only on freshly isolated BECs (57,3 ± 7,4%), while freshly isolated LECs were almost completely negative for CD146 expression (2,6 ± 0,7%). n = 4 independent skin donors. B In vitro cultured HDMECs at passage 1 (P1) were stained for CD146, CD31 and Prox1. Two subpopulations of BECs (CD31⁺/ Prox1⁻) and LECs (CD31⁺/ Prox1⁺) are identified in HDMECs (panel I). CD146 expression is detected on a small fraction of CD31⁺ cells (panel II), while Prox1⁺cells lack the expression of CD146 (panel III). Triple immunofluorescence staining shows the expression of CD146 only in CD31⁺Prox1⁻ cells, corresponding to BECs (panel IV). Cell nuclei are stained with Hoechst (blue). Scale bar: 100 μm. (n = 3 independent donors). C Representative immunofluorescence images of BECs and LECs at passage 1 separated by FACS and cultured in vitro. CD31 and Prox1 staining confirm the purity of the two cell populations. Whereas CD146 expression is detected only on BECs, LECs lack the expression of CD146. Cell nuclei are stained with Hoechst (blue). Scale bar: 100 μm. (n = 3 independent donors). D Western blot analysis of sorted BECs and LECs shows the expression of CD146 only on BECs. Detection of Prox1 only on LECs confirmed the purity of the populations. Fibroblasts (FBs) were used as negative control. Equal loading was assessed with anti-GAPDH antibody. (n = 3 independent donors)