Characterization of immunosorted NCAM1+ subpopulation A

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Characterization of immunosorted NCAM1+ subpopulation A Characterization of immunosorted NCAM1+ subpopulation A.RT‐PCR analysis of gene expression in NCAM1 cell fractions. Characterization of immunosorted NCAM1+ subpopulation A.RT‐PCR analysis of gene expression in NCAM1 cell fractions. GAPDH was used as endogenous control and NCAM1− cell were used as the calibrator sample for RQ calculation (therefore = 1). Data were analysed using SDS 3.2 software and presented as average RQ ± SDEV of three replicates. ***p < 0.001, *p < 0.05 versus NCAM1−.B–D.Immunofluorescence staining of NCAM1+ and NCAM1− subpopulations for E‐cadherin (E‐cad) (B, red) vimentin (C, green) and SIX2 (D, green). Nuclei stained with Dapi (blue). (B–C) Images were obtained using Olympus DP72 camera attached to Olympus BX51 fluorescence microscope and processed via cellSens standard software, bar represents 200 μm. (D) Images were obtained using Zeiss LSM 510 confocal microscope, bar represents 50 μm.E.Fluorescent quantification of SIX2 immunostaining as represented in (D).F.Double labelling of sorted NCAM1+ cells for NCAM1 and SIX2: NCAM1 with DAPI (upper panel; red and blue channels), NCAM1 with SIX2 (lower panel; red and green channels), indicating both NCAM1+ SIX2+ (arrows) and NCAM1+ SIX2− (arrowheads) cells.G.Graph represents percentage of NCAM1+ SIX2+ cells and NCAM1+ SIX2− cells.H.Clonogenic efficiency of NCAM1+ cells sorted from hFK and cultured in SFM. Data are presented as average CE(%) ± SDEV. **p < 0.01 versus NCAM1−.I.Representative morphology of NCAM1+ and NCAM1− clones. Cells were observed using a Nikon Digital Sight camera attached to a Nikon Eclipse TS100 microscope.J.Clonogenic capacity of hFK cells treated with IMGN901(ADC 55 nM), huN901 (Ab55 nM) or not treated (control). Data are presented as average CE(%) ± SDEV. ***p < 0.001, *p < 0.05 versus control group. Orit Harari‐Steinberg et al. EMBO Mol Med. 2013;5:1556-1568 © as stated in the article, figure or figure legend