Elution buffer

5–10×10⁶ RD cells were plated as spheres for 24–48 hours. Spheres were then cross-linked with 1% formaldehyde for 10–15 min, quenched with 0.125M glycine for 10min, and washed with PBS. Cells were resuspended in lysis buffer (50mM Tris pH7.5, 150mM NaCl, 5mM EDTA, 1% Triton-X, 0.1% SDS, 0.5% sodium deoxycholate) and sonicated (Misonix XL-2000) for 14 cycles (12 sec on, 2 min off). Cell debris was pelleted, and chromatin was precleared with Protein G agarose beads (Millipore) for 2 hours at 4°C. RBPJ antibody (Cell Signaling #5313) was added at 1:50 and rotated overnight at 4 °C. Protein G beads were added the next day for 3 hours with rotation at 4 °C. Beads were washed according to the Abcam protocol and DNA was eluted with elution buffer (Santa Cruz) at 67°C for 2 hours with rotation. Crosslinks were reversed overnight followed by a proteinase K digestion for 1hr. DNA was purified using the Qiagen PCR Purification kit. ChIP enrichment was evaluated using semi-quantitative PCR followed by quantitation using ImageJ (NIH) similar to previous published work (57 (link)). ChIP primers are listed in Supplementary Table 1.

Following growth in tumor-conditioned medium for the time s indicated in the figure legends, DC progenitor cells were harvested, protein and DNA were cross-linked with formaldehyde, and DNA was sheared by sonication. Equal amounts of chromatin [based on protein content as determined by BCA protein assay (Pierce)] were incubated with antibodies against STAT1, STAT3, or STAT5 or with an IgG isotype control antibody (all from Santa Cruz Biotechnology), and were immunoprecipitated with recombinant-protein A/agarose beads (Repligen). Cross-linking was reversed by overnight incubation at 65°C and elution with elution buffer (Santa Cruz Biotech). DNA binding was measured by real-time SYBR green PCR (iScript cDNA synthesis, Bio-Rad) performed with promoter-specific primers for each gene of interest (see table S2) with an AB7900HT real-time qPCR machine (Applied Biosystems). Data are presented as fold change in the abundance of the promoter (based on the number of PCR cycles needed to reach the threshold of detection) immunoprecipitated by the specific antibody compared to the control IgG, normalized to KG1 cells cultured in normal medium.