Fig 2: MAD2 modulates pluripotency and stemness ability in GC cell lines. (A) The interference of MAD2L1 in MKN45CSCs (shM) was confirmed by RT-qPCR. The relative expression levels of NANOG, SOX2, OCT3/4, LGR5, CDH1, VIM and LOXL2 mRNAs were quantified by RT-qPCR in the MKN45CSCs. Each gene was normalized to GAPDH. Fold changes were calculated relative to the non-interfered CSCs (WT). At least, three independent experiments were performed with similar results. (B) Flow cytometric analysis of CXCR4, CD90 and CD133 cell surface markers in MKN45CSCs after interference of MAD2L1. The histograms summarize the percentage of CXCR4-, CD90- and CD133-positive cells in sphere cultures from three different experiments. Fold changes were calculated compared to WT. Representative plots with the percent-positive cells present within the single-cell, live and debris-free population are shown. (C) ALDH activity profile. MKN45CSCs after interference of MAD2L1 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. Representative plots are shown, in the presence or absence of the ALDH inhibitor (DEAB), with the percent-positive cells present within the single-cell, live and debris-free population. (D) Secondary sphere formation efficiency in MKN45CSCs with reduced MAD2 levels. 1 cell/well was seeded by sorting and spheres were counted 10 days later. Graphs show the percentage of the total number of spheres formed/total number of cells seeded (SFE: sphere formation efficiency). Right panels show representative images of tumorspheres. Scale bar: 100 µm. (E) RT-qPCR for MAD2L1 and ALDH activity profile in SNU638CSCs. mRNA levels of MAD2L1 and frequency of ALDH-positive cells were quantified as in (A) and (C), respectively. (F) Secondary sphere formation assay in SNU638CSCs with reduced MAD2 levels. 250 cells/well were seeded and SFE was calculated as in (D). Panels below show representative pictures of tumorspheres. Scale bar: 100 µm. (G) Secondary sphere-forming capacity of MKN45CSCs in presence of the MAD2 inhibitor, M2i-1 (25 µM), after ten days, 1 cell/well in left panel (by sorting) and 5 cells/well in right panel. Graphs show the percentage of the total number of spheres formed/total number of cells seeded. Right panels show representative images of tumorspheres when seeding 5 cells/well. Scale bar: 100 µm. The experiments (D, E, F, G), were repeated three times with similar results. Statistical significance was evaluated with two-tailed Student's t-test (*P < 0.05; **P < 0.01; ***P < 0.001).