PMC

Failure-free survival and GLI1/18S expression (top). Overall Survival and GLI1/18S expression (bottom).

GLI1 and PTCH RNA levels were measured in triplicate by qRT-PCR with an 18S rRNA internal control.

The ratios are depicted in box plots. The median GLI1/18S ratios found are depicted by bars within the squares: ARMS 0.0014, ERMS 0.02, and US 0.0075. The boxes represent the 25^th and 75^th percentile of GLI1 expression. Standard deviation error bars are shown. Dots represent the distribution of GLI1 tumor expression within each tumor type.

The levels of GLI1 (A) and PTCH (B) mRNA were measured by RNase protection assay (RPA) using GAPDH as an internal control. The relative mRNA levels were extrapolated from a standard curve generated during the run. RPA expression of GLI1 was validated by qRT-PCR (C).

Using qPCR, the relative GLI1 copy number was determined in comparison to MGP, an unrelated gene on the opposite arm of chromosome 12. The methodology was validated by comparison to results obtained by Southern methodology. Standard curves were generated from analysis of DNA from normal lymphocyte (229) and the RH30-A cell line.

GLI1 and PTCH RNA levels were measured in triplicate by qRT-PCR with an 18S rRNA internal control. The expression of GLI1/18S and PTCH/18S RNA in ERMS tumors is depicted in box plots. The boxes represent the 25^th and 75^th percentile of GLI1 or PTCH expression. The median GLI1/18S ratio represented by the bars within the boxes was 0.013, 0.03, and 0.0035 for high, intermediate, and low GLI1 expressing tumors, respectively. For the same categories of GLI1 expression, PTCH/18S expression was 0.035, 0.0092, and 0.0054. Standard deviation error bars are shown. Dots represent the distribution of GLI1 and PTCH expression within each ERMS subset.

Ingenuity Pathways Analysis was used to identify genes downstream of Hh that were differentially expressed between high + intermediate versus low GLI1. Molecules are represented as nodes, and the biological relationship between two nodes is represented as an edge (line). All edges are supported by at least one reference from the literature. The intensity of the node color indicates the degree of up- (red) or down- (green) regulation. Genes depicted in gray were not differentially expressed in a statistically significant manner (p > 0.05). Nodes are displayed using various shapes that represent the functional class of the gene product.