These three regions contain consensus binding sequences for several transcription factors including AP2, PUF, STF, PEA3, E2F and PEA3. We analyzed the Cyclin D2 promoter by two indepen dent methods. The first was MCA Meth. The second method was MSP PCR. Unmethylated primers amplified the selleck chemicals region from 1616 to 1394 giving a product of 222 bp. methylated pri mers amplified the region from 1394 to 1152 producing a pro duct of 276 bp. PTCH1 promoter methylation analysis We analyzed the promoter region of PTCH1 from 238 to 62 bp, which corresponds with exon 1B. To assess methylation of the PTCH1 promoter, we fol lowed the MCA MSP method. This method gives us two melting peaks which are dependent on amplifica tion with unmethylated or methylated primers. Statistical analysis Statistical analyses were performed using Graphpad Prism 4.

Data graphed with error bars represent mean and SD from experiments performed in triplicate, unless otherwise noted. Fishers exact test was used to determine the sig nificance of any difference. Results Expression of GLI1 target genes after siRNA mediated GLI1 silencing We transfected Daoy and U87MG cell lines with GLI1 siRNA and scrambled siRNA. We monitored the effi ciency of transfection using lipofectamine through the delivery of the BLOCK iT Fluorescent Oligo. Images were captured using a fluorescent ima ging microscope and similar efficiencies of transfection were attained for both the GLI1 siRNA and the scrambled siRNA. To assess efficiency of silencing, expression of GLI1 was monitored by RT PCR after transfection.

Approximately 86% and 40 4 5% silencing of GLI1 transcript was achieved in Daoy and U87MG cell lines respectively. Silencing of GLI1 resulted in a 50% and 60% decrease in PTCH1 expression in Daoy and U87MG cell lines respectively. In contrast, Cyclin D2 showed a 17% decrease in Daoy and 113% increase in U87MG cells. Plakoglobin expression was decreased by 30% in Daoy cells and increased by 125% in U87MG cells. PAX6 expression was decreased by 35% in Daoy cells and increased by 100% in U87MG cells, and NKX2. 2 was decreased by 50% in Daoy cells and unaltered in U87MG cells. All changes listed were specific to GLI1 silenced cells and cells transfected with scrambled siRNA or untransfected cells did not show similar trends.

PTCH1 siRNA mediated silencing of GLI1 resulted in an 86% decrease in GLI1 transcript and a 50% decrease in PTCH1 transcript in the Daoy cell line compared with scrambled siRNA and untransfected Daoy cell lines. We subsequently sought to determine the pattern of PTCH1 transcript expression in 6 medulloblastoma cell lines and 14 primary medulloblastoma samples and observed that 50% of the cell lines and tumor samples showed high expression of PTCH1. Carfilzomib However, we were unable to find any significant correla tion between GLI1 and PTCH1 expression in the cell lines and tumor samples.