Supplementary Figure 3: OSMR is a direct target gene of STAT3

(a-b) STAT3 Kaplan-Meier survival plots were generated for glioblastoma patients with elevated STAT3 levels using the TCGA or REMBRANDT database, as described in the methods section. (c-d) Multivariate analysis with patient age, IDH mutation status and tumor grade as covariates were performed using two main analytical approaches of Cox proportional hazards (left panel) and Kaplan-Meier stratified logranks (right panel). OSMR and STAT3 are important predictors of survival even after controlling for IDH1 mutation, age, and tumor grade. Abbreviations are: O, OSMR; T, tumor grade; A, age; I, IDH1 mutation; S, STAT3; O|T,A,I: correlation of OSMR upregulation to survival after taking into consideration tumor grade, age, IDH1 mutation; S|T,A,I: correlation of STAT3 upregulation to survival after taking into consideration tumor grade, age, IDH1 mutation as co-variants. (e) STAT3 ChIP-Seq peak obtained at the Osmr promoter is shown. The peak and the STAT3 motif within the peak are both highly conserved across different species. (f) Protein-DNA complexes of EGFRvIII-expressing astrocytes were subjected to immunoprecipitation with a STAT3 antibody or rabbit IgG control. Immunoprecipitated DNA was analyzed by RT-qPCR using two different ChIP primer pairs (OSMR-a, OSMR-b) designed to amplify around the peak summit. An unknown region of chromatin 5 was used as negative control. (g) Lysates of MSCV; Stat3^loxP/loxP, EGFRvIII; Stat3^loxP/loxP, MSCV; Stat3^-/-, and EGFRvIII; Stat3^-/- astrocytes were subjected to immunoblotting using OSMR and STAT3 antibodies. Tubulin was used as loading control. (h) mRNA obtained from EGFRvIII; Stat3^loxP/loxP and EGFRvIII; Stat3^-/- was analyzed by RT-q-PCR to validate 28 randomly selected target genes identified in the RNA-Seq analyses. Fold change was normalized to GAPDH. OSMR is among validated target genes that are highly expressed in EGFRvIII; Stat3^loxP/loxP relative to EGFRvIII; Stat3^-/- (i) siRNA knockdown of STAT3 was induced in EGFRvIII-expressing human BTSC73 and the RNA of BTSC73-STAT3-KD and corresponding scramble control were subjected to RT-q-PCR analyses using primers for STAT3 and OSMR. Fold change expressions were normalized to GAPDH.