Article Figures & Data
Figures
- Figure 1.
Murine astrocytes harvested from the cerebral hemispheres and stained for GFAP.
- Figure 2.
A, MGMT gene expression in wild-type and knockout p53 murine astrocytes. RNA was isolated from wild-type and knockout astrocytes and RT-PCR was done to assess MGMT gene expression. MGMT expression was significantly higher in wild-type than in knockout p53 astrocytes. Murine β2-microglobulin was used to control for the PCR reaction. B, MGMT protein expression in wild-type and knockout p53 murine astrocytes. Protein extracts were prepared from wild-type and knockout astrocytes and expression of MGMT was analyzed by Western blot. Actin was used as a loading control. MGMT protein levels were higher in wild-type than in knockout p53 astrocytes.
- Figure 3.
RT-PCR to examine the effect of p53 knockdown on MGMT expression in wild-type p53 astrocytes. Cells were treated with a control oligo (CNTRL) or RNAi specific for p53 (RNAi). p53 knockdown was successful and accompanied by a significant reduction in MGMT gene expression. Murine β2-microglobulin was used to control for the PCR reaction.
- Figure 4.
A, MGMT promoter methylation analysis in wild-type and knockout p53 murine astrocytes. DNA was extracted from wild-type and knockout p53 astrocytes. After bisulfite modification, methylation-specific PCR for the MGMT promoter was done. In both wild-type and knockout p53 astrocytes, the MGMT promoter is unmethylated. Unmethylated MGMT promoter (U), methylated MGMT promoter (M). B, MGMT promoter examined for a p53 consensus site. Two previously unidentified p53 half sites are highlighted. C, CHIP assay to examine the interaction of p53 with the MGMT promoter. IgG controlled for the immunoprecipitation component of the assay and a region of DNA ∼5,000 bp downstream of the MGMT promoter was controlled for nonspecific p53-DNA interactions (CNTRL). p53 was detected at the first intron of the MGMT gene promoter.
- Figure 5.
A, RNAi knockdown of p53 in SF767 human astrocytic glioma cells. Cells were treated with RNAi specific for p53 and RT-PCR was done to examine MGMT gene expression. Cells in which p53 expression was inhibited (RNAi) expressed lower levels of MGMT than cells treated with a control duplex (CNTRL). B, MGMT promoter methylation analysis of SF767 cells. After p53 knockdown, bisulfite modification and methylation-specific PCR were done. The methylation status of the MGMT promoter in SF767 cells was unaffected by knockdown of p53. Unmethylated MGMT promoter (U), methylated MGMT promoter (M).
Volume 67, Issue 2
