Characterization of phenotypic features of SP and non-SP cell fractions from parental tumorigenic and invasive WPE1-NB26 cells by the Hoechst dye efflux technique and FACS.

<p><b>a</b>) Representative data of the Hoechst dye efflux profile obtained after staining of parental WPE1-NB26 cell line with fluorescent Hoechst dye showing the SP cell subpopulation (green) and non-SP fraction (blue) detected in the total mass of parental WPE1-NB26 cells. <b>b</b>) FACS profiles obtained after staining of parental WPE1-NB26 cells with phycoerythrin -labeled anti-CD133 antibody showing the percentage of CD133⁺ and CD133⁻ PC cells detected in the total mass of parental WPE1-NB26 cells. <b>c</b>) Comparative Western blot analyses of expression levels of prostatic stem cell-like markers (CD133 and CD44), multidrug transporter ABCG2, EGFR, Ser⁴⁷³-pAkt, NF-κB p65 subunit and secreted MIC-1 proteins detected in the SP and non-SP cell fractions isolated from parental WPE1-NB26 cell line. <b>d</b>) Immunofluorescence staining of methanol-fixed prostaspheres derived from SP cells and the adherent non-SP cell fraction isolated from the parental WPE1-NB26 cell line were done with anti-EGFR plus Tyr¹¹⁷³-pEGFR, Ser⁴⁷³-pAkt, NF-κB p65 or MIC-1 primary antibody plus fluorescein (green) and/or Texas red secondary antibody (red) and 4′,6-diamidino-2-phenylindole (nuclear blue) after blocking with goat serum. Representative pictures showing the expression level and cellular localization obtained for the stem cell-like markers including CD133 (red), CD44 (green) and ABCG2 (red) as well as overlaps of EGFR/Tyr¹¹⁷³-pEGFR (red/green, hybrid yellow), Ser⁴⁷³-pAkt (red), NF-κB p65 (red) and MIC-1 (red) are shown at the original magnification of ×630.