Extended Data Fig. 4: CCDC25 binds to NET-DNA.
From: DNA of neutrophil extracellular traps promotes cancer metastasis via CCDC25
a, Schematic of NET-DNA pull-down assays. b, Mass spectrometry analysis identified CCDC25 as the cytoplasmic membrane protein from MDA-MB-231 cells pulled down by the biotinylated NET-DNA. c, Immunoblotting of the membrane proteins of MDA-MB-231 cells pulled down by biotinylated NET-DNA and detected by an anti-CCDC25 antibody. d, EMSA demonstrated NET-DNA binding to CCDC25 super-shifted by an anti-CCDC25 antibody. Membrane proteins of MDA-MB-231 cells and the biotinylated NET-DNA were incubated with or without the antibody against CCDC25, IgG (negative control), or 20-fold excess of unbiotinylated NET-DNA. e, The binding of NET-DNA to the membrane proteins of MDA-MB-231 cells transduced with a control sgRNA or two CCDC25-sgRNAs was evaluated by EMSA. f, EMSA reveals the interaction of biotinylated NET-DNA with increasing concentrations of CCDC25. The protein–DNA complex is denoted by a red asterisk. g, Binding kinetics of CCDC25 and NET-DNA generated from the above EMSA assays in f. n = 3. Data are mean ± s.d. h, Purified NETs were coupled to magnetic beads, treated with Proteinase K (left) and DNase I (right), and incubated with His–CCDC25. The interaction of NETs and CCDC25 was evaluated by the precipitation of NETs–beads and blotted with anti-His antibody. His–CCDC25 mixed with beads without DNA served as a negative control (empty beads). The digestion efficiency of the protein and DNA components of NETs by Proteinase K and DNase I was confirmed by immunoblotting for H3cit and agarose gel analysis for DNA. i, j, Three different biotinylated heterologous 90-bp DNA duplexes with random sequences were either irradiated with UV-C light or were not irradiated. i, The relative 8-OHdG content in the DNA was determined by ELISA. n = 3. Data are mean ± s.d., **P = 0.0059 as determined by a two-tailed Student’s t-test. j, DNA pull-down assay for His–CCDC25. The resultant CCDC25 was detected by anti-His western blot analysis. His–CCDC25 mixed with beads without DNA served as a negative control (empty beads). k, Representative bio-layer interferometry showing CCDC25 binding to 8-OHdG-enriched DNA (left) and non-8-OHdG-enriched DNA (Right). The coloured lines show the data for five different concentrations of CCDC25 as indicated. Data in b–f, h–k are representative of three biologically independent experiments.
