Table 1.
Primer sequences used to analyze MMR.
Fig 1.
A) Relative growth rates (RGR) for tumor cell lines treated with TMZ. B) Relative growth rates for tumor cell lines before and after TMZ cycling treatment (B: basal cells; 1C: first TMZ cycle; 2C: second TMZ cycle). SF268 and SK-N-SH lines did not reveal any IC50 variation, whereas A172 and LN229 showed a large increase in TMZ IC50. All data represent the mean value ± SD of triplicate cultures.
Fig 2.
Cell cycle modulation induced by TMZ treatment.
Modulation of the cell cycle (G1, S and G2) by TMZ treatment in A172 (A), LN229 (B), SF268 (C) and SK-N-SH (D) cell lines. The number of cells in the G1-, S or G2-phase is given as percentages of the total cell population.
Fig 3.
Analysis of MGMT promoter methylation and MGMT expression levels.
A) Modulation of the MGMT promoter methylation percentage in tumor cell lines before and after TMZ treatment. B) Real-time PCR analysis of MGMT expression before and after TMZ treatment. Showing relative expression of MGMT compared with SK-N-H cell line. C) Western blot analysis of MGMT protein expression in tumor cell lines before and after TMZ treatment. Beta-actin expression was used as a control. The graphs on the right show the densitometry of the MGMT Western blot analysis in SF268 and SK-N-SH cell lines before and after TMZ treatment. B: basal cells; 1C: first TMZ cycle; 2C: second TMZ cycle. All data represent the mean value ± SD of triplicate cultures.
Fig 4.
Modulation of MGMT expression by 5-Aza.
A) Densitometric analysis of the effect of 5-Aza for each cell line showing relative expression of MGMT compared with beta-actin under each experimental condition. Western blot analysis of MGMT expression after 5-Aza exposure in tumor cell lines. Beta-actin expression was used as a control. B) Relative growth rates of tumor cell lines before and after 5-Aza exposure. B: basal cells, 5Aza: cell line treated with 5-Aza. All data represent the mean value ± SD of triplicate cultures.
Fig 5.
Modulation of MGMT expression by O6-BG in SF268 and SK-N-SH tumor cell lines.
A) Western blot analysis of the MGMT expression after treatment with TMZ, O6-BG and both TMZ + O6-BG. B) Relative growth rates of SF268 and SK-N-SH cell lines with and without O6-BG treatment after TMZ treatment. B: basal cells, O6BG: cell line treated with O6-BG. All data represent the mean value ± SD of triplicate cultures.
Fig 6.
Modulation of MMR expression by TMZ treatment.
A) Real-time PCR analysis of MMR gene expression levels (MLH1, MSH2, MSH3, MSH6 and PMS2) in tumor cell lines. B) Modulation of MMR gene expression levels after TMZ treatment in tumor cell lines. B: basal cells; 1C: first TMZ cycle; 2C: second TMZ cycle. All data represent the mean value ± SD of triplicate cultures.
Fig 7.
Modulation of P-gp expression by TMZ treatment.
A) Real-time PCR analysis of P-gp expression levels in tumor cell lines. The TMZ-resistant HCT15 cell line was used as a control. B) Modulation of P-gp expression by TMZ treatment in tumor cell lines. C) Relative growth rates after exposure to TMZ in tumor cell lines before and after verapamil (V) treatment. B: basal cells; 1C: first TMZ cycle; 2C: second TMZ cycle. All data represent the mean value ± SD of triplicate cultures.
Fig 8.
Modulation of CD 133 expression by TMZ treatment.
A) Flow cytometry analysis of percentages of CD133 positives in tumor cell lines using TMZ treatment B: basal cells, 1C: first TMZ cycle; 2C: second TMZ cycle. Data were calculated from three independent experiments. B) Representative flow cytometry data for CD133 expression. SSC: side-scattered light.