Figure 2.

Characterization of GCSCs. (A) Relative levels of mRNA transcripts for pluripotency and stemness markers. The relative expression levels of NANOG, SOX2, OCT3/4, LGR5 and LOXL2 mRNAs were quantified by RT-qPCR in MKN45, ST2957 and SNU638 cell lines cultured in adherence (ADH) or as spheres (CSCs). Each gene was normalized to GAPDH. Fold changes were calculated and compared to ADH (set as 1.0). At least three independent experiments were performed with similar results. (B) Flow cytometric analysis for CXCR4, CD44, CD90 and CD133 cell surface expression and autofluorescence (Fluo) in the 3 GC cell lines cultured in adherence (ADH) or as spheres (CSCs). The histograms summarize the percentage of CXCR4-, CD44-, CD90-, CD133- and autofluorescent-positive cells from three different experiments. Fold changes were calculated compared to ADH (set as 1.0). (C) ALDH activity profile. MKN45, ST2957 and SNU638 cell lines cultured in adherence (ADH) or as spheres (CSCs) and were counter-stained with AldeRed to determine the frequency of ALDH-positive cells by flow cytometry, in the presence or absence of the ALDH inhibitor (DEAB). ALDH activity is expressed as mean of the percentage of positive cells of 3 experiments. (D) Activation of signaling pathways in CSCs. Representative Western Blot analysis for GLI1, cleaved Notch1 and β-catenin in the 3 GC cell lines cultured in adherent and sphere conditions. Graphs show the fold change of protein levels compared to ADH expression obtained in three different experiments performed under the same conditions. Protein levels were normalized with α-tubulin. The statistical significance was evaluated with two-tailed Student's t-test (*P < 0.05; **P < 0.01; ***P < 0.001).