Cells were lysed in 1× radioimmunoprecipitation assay (RIPA) buffer (Cell Signaling Technology) supplemented with protease inhibitor cocktail (Sigma-Aldrich), followed by brief sonication, and the lysate was cleared by centrifugation at 20,000g for 10 min. For cytosol/nuclear fractionation, cells were incubated in buffer A (10 mM Hepes-KOH at pH 7.5, 50 mM NaCl, 500 mM sucrose, 0.1 mM EDTA, 1.5 mM MgCl2, and protease inhibitor cocktail) for 15 min, followed by mixture with Triton X-100 (0.5% at final) for 1 min, and the cytosol fraction was cleared by centrifugation at 16,000g for 5 min. The nuclear pellet was briefly washed in buffer B (15 mM Hepes-KOH at pH 7.5, 60 mM KCl, 15 mM NaCl, 0.32 mM sucrose, and protease inhibitor cocktail), dissolved in 1× RIPA buffer, and briefly sonicated, and the nuclear fraction was cleared by centrifugation at 20,000g for 10 min.

For coimmunoprecipitation, the nuclear pellet from cells expressing S100A8-Flag was dissolved in buffer C (50 mM tris-HCl at pH 8.0, 420 mM NaCl, 1.5 mM MgCl2, 0.4% NP-40, 25% glycerol, and protease inhibitor cocktail) for 1 hour, diluted to 100 mM NaCl strength with buffer D (50 mM tris-HCl at pH 8.0, 0 mM NaCl, 1.5 mM MgCl2, 0.4% NP-40, 25% glycerol, and protease inhibitor cocktail), and treated overnight with Benzonase (Millipore), and the nuclear extract was cleared by centrifugation at 20,000g for 10 min. Coimmunoprecipitation and Western blot analyses were performed as previously described (46).

For imaging analysis, cells were fixed with 4% paraformaldehyde in PBS, and immunofluorescence staining was performed with rabbit anti-S100A9 and mouse anti-S100A8, followed by treatment with anti-rabbit immunoglobulin G (IgG)–fluorescein isothiocyanate and anti-mouse IgG–Texas Red. Clinical immunohistology data of S100A9 and S100A8 were obtained from The Human Protein Atlas database (47); for breast cancer, S100A9 staining data were from PatientID_2091, and S100A8 staining data were from PatientID_2392; for skin cancer, S100A9 staining data were from PatientID_2166, and S100A8 staining data were from PatientID_2150.

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