Fig 2: Secretion of IL-4 in CD133+ SP cells. (A) Western blot analysis showed elevated secretion of IL-4 in CD133+ SP cells, compared with MP cells. (B) Relative expression levels of IL-4 compared with GAPDH in SP and MP cells, from the western blot analysis. (C) Cell viability of the CD133+ SP cells was significantly reduced following pretreatment with anti-IL-4 for 24 h. (D) Tumor spheres generated by CD133+ SP cells became more sensitive to apoptosis following pretreatment with anti-IL-4 for 24 h. Magnification, ×25. Data are presented as the mean ± standard error of the mean. *P<0.05 and **P<0.01, between SP and MP cells. IL-4, interleukin-4; CD, cluster of differentiation; SP, side population; MP, main population; 5-FU, 5-fluorouracil.

Fig 3: CD133+ SP cells show high levels of differentiation. (A) In vitro proliferation assay revealed that the proliferation rate of the CD133+ SP cells were significantly higher, compared with the MP cells. (B) Morphology of the CD133+ SP cells changed rapidly on day 5 and later developed filaments, which resembled fibroblast. Magnification, ×100. Data are presented as the mean ± standard error of the mean. *P<0.05 and **P<0.01, between SP and MP cells. CD. cluster of differentiation; SP, side population; MP, main population; OD, optical density.

Fig 4: CD133+ SP cells exhibit high self-renewal capacity. (A) A clone formation efficiency assay revealed that the total number of tumor spheres generated by the CD133+ SP cells were significantly higher, compared with the number generated by the MP cells. (B) Quantification of the results of reverse transcription-quantitative polymerase chain reaction analysis showed that the relative mRNA expression levels of Oct-4, EpCAM, Sox-2, Bmi-1 and Nestin were significantly upregulated in the CD133+ SP cells, compared with the MP cells. (C) Fluorescence microscopy revealed that the CD133+ SP cells exhibited more positive CD44 fluorescence and EpCAM stem cell proteins, whereas this fluorescence was not enriched in the MP cells. Magnification, ×400. Data are presented as the mean ± standard error of the mean. **P<0.01, vs. SP. Oct-4, octamer-binding transcription factor-4; EpCam, epithelial cell adhesion molecule; Sox-1, (sex determining region Y)-box 2; Bmi-1, B-cell-specific Moloney murine leukemia virus insertion site-1; CD. cluster of differentiation; SP, side population; MP, main population.

Fig 5: CD133+ SP cells are highly tumorigenic and invasive. (A) CD133+ SP cells initiate tumor growth in NON/SCID mice significantly faster, compared with MP cells. (B) Immunohistochemistry showed that SP cell-derived tumor tissues were more positive to the CD133 stem cell protein. Magnification, ×250. (C) CD133+ SP cell invasiveness was measured using a Matrigel assay. The number of CD133+ SP cells, which invaded across the membrane were significantly higher, compared with the number of MP cells. Data are presented as the mean ± standard error of the mean. **P<0.01, vs. SP. CD, cluster of differentiation; SP, side population; MP, main population.