Supplementary Figure 1: Gene expression profiling of EGFRvIII-expressing human brain tumor stem cells (BTSCs) and mouse astrocytes

(a) The protein lysates of human BTSCs were analyzed by immunoblotting using the antibodies indicated on the blots. Wild type EGFR and EGFRvIII bands are marked with * and **, respectively. (b) Correlation of EGFRvIII expression with OSMR or phosphorylated STAT3 (p-STAT3-Ser-727) was obtained by running Pearson analyses on the densitometric values of protein expression normalized to tubulin. (c) Three EGFRvIII-expressing BTSCs (# 68, 73, and 90) and a BTSC that did not express EGFRvIII (# 41) were used for gene expression profiling by RNA-seq analyses. Differentially expressed genes in each of the BTSC68, 73, and 90 were called relative to BTSC41 control by Tophat/Cufflinks RNA-seq analysis pipeline. Hierarchical clustering was performed to group genes with similar expression patterns in at least two EGFRvIII-expressing BTSCs relative to control in each pair-wise comparison and the heat map plot was generated to show the expression ratios of the genes that are differentially expressed [fold change (fc) > 5; p value <10⁻⁵]. Yellow and blue colors indicate upregulated or downregulated genes in EGFRvIII-expressing lines, respectively. (d) Among candidate target genes that were upregulated in an EGFRvIII dependent manner based on RNA-seq analyses, 10 randomly selected genes and OSMR were validated by RT-q-PCR in EGFRvIII-expressing BTSC73 versus three different BTSC control lines that do not express EGFRvIII (#12, 41, and 139). Gene expression was normalized to GAPDH. The graph represents downregulation of these genes in control lines relative to BTSC73. (e) EGFRvIII-expressing or MSCV-infected control astrocytes that express STAT3 (Stat3^loxP/loxP) or in which STAT3 is conditionally deleted (Stat3^-/-) were subjected to RNA-seq analyses as described for human BTSC. These are labeled EGFRvIII; Stat3^loxP/loxP, EGFRvIII; Stat3^-/-, MSCV; Stat3^loxP/loxP and MSCV; Stat3^-/- on the heat map. The heat map plot (top panel) shows the genes that are downregulated (417 genes, fc ≤ 2) in EGFRvIII; Stat3^-/- compared to EGFRvIII; Stat3^loxP/loxP. Most of these 417 genes are unchanged between MSCV; Stat3^loxP/loxP and MSCV;Stat3^-/-. The heat map plot (lower panel) shows the genes that are upregulated (47 genes, fc ≥2) in EGFRvIII; Stat3^-/- compared to EGFRvIII; Stat3^loxP/loxP. Red color indicates highly expressed genes and white color indicates low expression.