Figure 5

Epigenetic silencing of the Rb promoter in glioma cells. A, Western blot of the Rb protein in four different glioma cell lines and erythroleukemic K562 cells. The blot was re-incubated with a human β-actin antibody for normalization and the resulting data were plotted (right panel). B, RT-PCR assay using radioactive duplex-PCR for semi-quantitative transcription level determination was performed. The results of four independent experiments are plotted and standard errors are shown. C, DNA methylation status of the U87MG glioma cells was determined by sodium bisulfite sequencing. D, Chromatin immunoprecipitation assay from U87MG glioma cells, HeLa cells and human quiescent lymphocytes. As a positive control, the in vivo CTCF binding at the Imprinting Control Region of the Igf2/H19 (Igf2-ICR) imprinted locus is shown in U87MG glioma cells. Additional controls are shown using HeLa cells. As a negative control we used primers covering the Rb Exon 27 (546 bp product, with genomic position +175819 to +176365) located ~200 kb downstream of the Rb promoter. The linear range of amplification is shown as Input for the U87MG series of ChIPs, and IgG was used as a non-specific antibody.