Fig. 6.

CD271 regulates proliferation and adhesion via separate signaling axes. a Representation of the lentiviral vector for stable overexpression of the Intra Cellular Domain (ICD) of CD271. The viral backbone contains Green Fluorescent Protein (GFP) and infrared fluorescent protein (iRFP) reporters under the SV40 and PGK promoters respectively, as well as the ICD overexpression cassette (ICD OE) or an empty vector (EV) under the CMV promoter. b FACS analysis for EdU incorporation in cells overexpressing ICD (ICD OE) compared to control cells (EV) (n = 3; P value ≤ 0.05). c In vivo imaging of Nude mice injected with cells carrying the empty vector (EV) or the ICD overexpressing vector (ICD OE). The signal is from the iRFP present in the lentiviral backbone. d Quantification of the signal obtained by IVIS for iRFP (three mice for a total of six injections were analyzed for each conditions; P value_m1 ≤ 0.05, P value_m2 ≤ 0.05). Error bars indicate S.E.M. e Brightfield (upper panel) and fluorescent micrographs (lower panel) of melanoma cells in culture 72 h after infection with the EV or ICD OE vector. Scale bars 100 µm. f Crystal violet staining of adherent cells (upper panel) and suspension cells, after re-attachment on fibronectin-coated plates (lower panel), carrying either the control vector (EV) or the ICD overexpressing vector (ICD OE). g Quantification of adherent and suspension cells after infection with EV or ICD OE constructs (n = 3, P value_adh.cells ≤ 0.05, P value_susp.cells > 0.05). Error bars indicate S.D. h FACS analysis for EdU incorporation in cells infected with CMVTOEV or CMVTOCD271 constructs and transfected with sicontrol or siTrk-A. One day after siRNA transfection the cells were treated with doxycycline (1 μg/ml) for 24 h. Cells were then pulsed with EdU for 30 min and collected for FACS analysis (n = 3; P value ≤ 0.05). i Crystal violet staining of suspension cells (forced to reattach on fibronectin-coated plates) infected with CMVTOEV or CMVTOCD271 and transfected with siControl (upper panel) or siTrk-A (lower panel). The cells were treated, as described in h. j Quantification of suspension cells (CMVTOEV or CMVTOCD271 infected) after siRNA transfection and doxycycline administration (as described in h) (n = 3, P value ≤ 0.05, P value ≤ 0.001). Error bars indicate S.D. All experiments done with cell line M010817