Fig. 3

CD271 overexpression impairs proliferation and adhesion. a Schematic representation of the lentiviral vector used to stably overexpress CD271 in melanoma cell lines. The viral backbone carries the Red Fluorescent Protein (RFP) and infrared fluorescent protein (iRFP) reporters under the SV40 and PGK promoters respectively, as well as the CD271 overexpression cassette (CD271 OE) or an empty vector (EV) under the CMV promoter. b FACS analysis for EdU incorporation in cells 72 h after infection with EV or CD271 OE vectors (n = 3; P value ≤ 0.05). c Upper panel: in vivo imaging of Nude mice injected with cells carrying the empty vector (EV) or the CD271 overexpressing construct (CD271 OE). The signal is from the infrared protein present in the lentiviral backbone. Lower panel: Immunofluorescence for CD271 protein in xenografts obtained by injection of melanoma cells containing the EV or the CD271 OE vector. Scale bars 100 µm. d Quantification of the signal from iRFP by IVIS at different time points (four mice for a total of eight injections were analyzed for both control and CD271 overexpression; P value ≤ 0.001). Error bars indicate S.E.M. e Brightfield and fluorescent micrographs of melanoma cells in culture 72 h after infection with the EV or CD271 OE vector. Scale bars 50 µm. f Crystal violet staining of melanoma cells 72 h after infection with the EV or CD271 OE vector after washing (upper panel) and 2 h after suspension cells reattached on FN plates (lower panel visualized as inverted, black and white micrographs). g Quantification of the suspension cells re-attached on fibronectin-coated plates. (n = 3, P value ≤ 0.001) h qRT-PCR for CD271 levels in melanoma cells infected with EV or CD271 OE in the adherent and in the suspension fractions (n = 3, P value ≤ 0.001). Error bars for g and h indicate S.D. i Western blot for CD271 levels in melanoma cells infected with EV or CD271 OE in the adherent and in the suspension fractions. j Crystal violet staining of suspension cells re-attached on fibronectin-coated plates 10 days after seeding. All experiments done with cell line M010817