Additional file 1: Figures S1–S9. of The epigenetic modifier JMJD6 is amplified in mammary tumors and cooperates with c-Myc to enhance cellular transformation, tumor progression, and metastasis

Figure S1. The genome locus coordinates and number of genes in the minimal region of chromosome 11 amplicon in MMTV-Myc mammary gland tumors. Figure S2. CGH analysis of two cell lines derived from MMTV-Myc mammary gland umors. A. Chromosomal gains and losses in Myc83 and 88CT1 cell lines. Arrow indicates the chromosome 11 amplicon in Myc83 cells consistent with the original CNV observations made in Fig. 1 for MMTV-Myc tumors. 88CT1 cells do not contain this amplicon. B. RT-qPCR validation of higher expression of three genes contained within the amplified region in Myc83 compared to 88CT1 cells. Figure S3. Analysis of cell death induced by glucose deprivation or etoposide treatment in cells with knock-down of 6 genes contained within the amplified region of chromosome 11. Analysis was performed as in Fig. 3 using cells with stable expression of shRNAs targeting the FBF1, Ube2o, Birc5, TK1, Sumo2, and Tnrc6c genes. Originally, 5 shRNAs for each gene were tested and shRNAs that achieved more than a 50 % downregulation of the targeted gene were selected for further analysis. Myc was induced in NMuMG cells expressing pBabe-MycERTM by the addition of 150 nM 4-OHT (MycON) for 24 h. Myc-induced cell death was tested after treatment with 100 μM etoposide for 20 h. Figure S4. Efficiency of JMJD6 knock-down. Western blot analysis of JMJD6 expression in NMuMG and Myc83 cell lines stably expressing two different shRNA vectors. Figure S5. Ectopic expression of LacZ-V5 and JMJD6-V5 in NMuMG cells. A. Cells were stained with anti-V5 antibody, followed by incubation with a secondary antibody conjugated with Alexa-568 and DAPI. The images show nuclear localization for JMJD6 and cytoplasmic localization for LacZ. B. Western blot analysis of cells with JMJD6 overexpression using anti-JMJD6 antibodies. Figure S6. JMJD6 does not alter c-Myc protein levels or localization of MycER™ fusion protein. A. Western blot analysis of NMuMG-MycER™ cells expressing LacZ or JMJD6 with anti-Myc antibody after addition of 4-OHT. Left panel shows MycER™ fusion protein and right panel shows endogenous c-Myc protein in cells expressing LacZ or JMJD6. B. Immunofluorescent staining (anti-c-Myc antibody) of NMuMG-MycER™ cells expressing JMJD6 after treatment with 4-OHT (MycON) or ethanol (MycOFF) for 24 h. Figure S7. Reverse transcription-qPCR analysis of JMJD6 and p19ARF expression in mouse MMTV-Myc mammary gland tumors. RNA isolated from mammary gland tumors was processed as described in the “Methods” section. The ΔCT values for JMJD6 and p19ARF were normalized to mouse Tbp housekeeping gene expression. Figure S8. JMJD6 does not bind to p16 promoter in NMuMG cells. Primers for mouse p16 gene promoter were used as a negative control after chromatin immunoprecipitation with anti-JMJD6 antibody described in Fig. 7. Figure S9. Wild-type JMJD6, but not its mutant form, suppresses Myc-induced apoptotic cell death. The experiments were performed as described in Fig. 4. The percentage of dead cells was measured by FACS analysis of cells stained with Annexin V and propidium iodide. (PPTX 2558 kb