Page gels

Custom oligonucleotides were purchased from Biomers (www.biomers.net) and purified by high-performance liquid chromatography. The DNA concentration was determined by measuring the absorbance at 260 nm with a micro-volume spectrometer (NanoDrop 2000). Each DNA duplex was assembled by mixing a stoichiometric quantity of the strands involved in the gapped and fully paired duplex in 1 × TE/Na buffer (10 mM Tris, pH 7.5, 0.1 mM EDTA, 150 mM NaCl). The final concentration was 10 μM for each strand. The oligo mixtures were cooled slowly from 90 °C to room temperature in 10 l water placed in a styrofoam box over 48 h to facilitate strand hybridization. In all, 10% non-denaturing PAGE gels (Biorad) run in 1 × TBE (pH 8.3, Tris-borate-EDTA) buffer were used to confirm the assembly of each duplex. The electrophoresis experiment presented in Fig. 1b was performed on the crude reactions. The desired DNA structures migrate as a single sharp band, suggesting that F-duplex and G-duplexes were properly formed. More details regarding the DNA sequences used for the assembly of F-duplexes and G-duplexes are given in Supplementary Method 1.

HEK293 cells were collected after a 24-h treatment, and histones were extracted using the Abcam Histone Extraction kit according to kit instructions (Cambridge, United Kingdom). Histone protein concentration was measured by Qubit Protein Assay from Thermo Fisher Scientific (Waltham, MA, United States). Protein (5 μg) was loaded onto 4–15% Bio-Rad Page Gels and transferred to 0.45 μm nitrocellulose (Bio-Rad, Hercules, CA, United States). The blots were incubated with H3K27ac primary antibody (1:1000; Abcam ab4729) overnight at 4°C and anti-rabbit IgG conjugated secondary antibody (1:5000, Santa Cruz Biotechnology Sc-2357) for 1 h at room temperature. The histone protein was normalized to total histone 3 (H3; Abcam ab1791) and quantified with ImageJ software. We tested significance by comparing the ratio of H3K27ac/H3 with a two-tailed Student’s paired t-test (p < 0.05).

DIC was carried out as previously published^{8 (link)}. In short, antibodies against either CD63+ (BD bioscience) or β1-integrin+ (BD bioscience) were bound to Dynabeads (Thermofisher) at 5 μg of antibody per 1 mg beads. Conjugated beads were then washed, and incubated with DKO-1 pre-cleared conditioned media overnight, agitating at 4 °C. Following incubation, beads were washed three times in DIC wash buffer, then re-suspended in 1 × RIPA buffer (ThermoFisher). Samples were then loaded onto PAGE gels (Biorad) for western blot analyses.
DIC against the P10 pellet (microvesicles) was carried out in the same fashion but the P10 pellet was resuspended in DIC wash buffer, and then incubated with conjugated beads.