Supp. Fig. Legends from SOCS3 Modulates the Response to Enzalutamide and Is Regulated by Androgen Receptor Signaling and CpG Methylation in Prostate Cancer Cells

Supplementary Fig. S1 Epigenetic regulation of SOCS3 in cell lines. A, SOCS3 mRNA was measured in AR positive prostate cancer cell lines after treatement with 10 mM 5-Aza-2'deoxycytidine for 72 hours. B, publicly available Bisulfite-Seq. data for the SOCS3 CpG island was analyzed using the UCSC genome browser (hg19) and the ENCODE HAIB Methyl RRBS data track. A region of the SOCS3 promoter that is found to be differentially methylated in cell lines and tissue samples was selected for further methylation specific qPCR analysis. Supplementary Fig. S2 AR positive cell lines do not express IL6 mRNA. IL6 mRNA expression was measured by qPCR during cultivation under normal growth conditions. Supplementary Fig. S3 Knock-down and overexpression efficiency of SOCS3 in LNCaP. SOCS3 expression in LNCaP was modulated by stable over-expression and knock-down with lentiviral vectors and the effect on SOCS3 mRNA was determined by qPCR after 72 hours of reatment with 5 ng/ml IL6. Supplementary Fig. S4 AR binding to DNA occurs in close proximity of several important components of the IL6 pathway. Publicly available ChIP-Seq data sets were analyzed to visualize AR binding sites, displayed as black rectangles. A, no AR binding sites could be found in close proximity of SOCS3, whereas a positive control, PSA/KLK3, displayed abundand AR binding. B, AR binding sites could be identified in close proximity of several genes (IL6, IL6R, IL6ST and JAK1) of the IL6 signaling pathway. Detailed analysis of the identified regions demonstrated only low basal AR binding in absence of ligand. Treatment with R1881 induced AR binding to the DNA in these regions and enzalutamide was able to revert this effect. Supplementary Fig. S5 SOCS3 is not regulated on post-transcriptional level by R1881. A luciferase reporter vector containing the coding sequence (CDS) and the 3'UTR of SOCS3 in the untranslated region after the luciferase stop codon was transfected into LNCaP cells. Treatment with 1 nM R1881 did not influence the reported expression, whereas, as a positive control, co-transfection with a shRNA targeting SOCS3 reduced the luciferase activity by more than 50 %. The firefly luc2 luciferase activity was normalized to a co-transfected renilla luciferase control vector. Supplementary Fig. S6 Knock-down and overexpression of SOCS3 is sustained after 3 weeks of combined IL6 and Enzalutamide treatment. SOCS3 expression in LNCaP was modulated by lentiviral over-expression and knock-down with a pool of 3 individual shRNAs targeting SOCS3 and the effect on SOCS3 mRNA was determined by qPCR after 3 weeks of combined IL6 and enzalutamide treatment.