Fig 1: M2 macrophages reduce the radiosensitivity of HPV-negative HNSCC by releasing HB-EGF. (A) Correlation analyses between the fraction of the γ-H2AX foci and the expression of HB-EGF. (B) Representative images of fluorescent staining. Immunofluorescence analyses revealing the expression of γ-H2AX foci in CAL27 and SCC90 cells with or without HB-EGF (20 ng/ml) 5 h after 4 Gy radiation; magnification, ×200 (green, γ-H2AX staining; blue, DAPI). (C) Fraction of CAL27 and SCC90 cells with the residual γ-H2AX foci (%) with or without HB-EGF (50 ng/ml). **P<0.01. (D) Immunofluorescence analyses of the γ-H2AX foci of M2 macrophages co-cultured with CAL27 or SCC90 and anti-HB-EGF antibody (3 µg/ml), and M1 macrophages co-cultured with CAL27 or SCC90 and HB-EGF (20 ng/ml); magnification ×200 (green, γ-H2AX staining; blue, DAPI). M2 macrophages reduce the radiosensitivity of HPV-negative HNSCC by releasing HB-EGF. (E) Number of the γ-H2AX foci of M1 and M2 macrophages co-cultured with CAL27. (F) Number of the γ-H2AX foci of M2 macrophages co-cultured with CAL27 cells with anti-HBEGF-neutralizing antibody. (G) Number of the γ-H2AX foci of M1 macrophages co-cultured with CAL27 cells with HB-EGF. Results are presented as mean ± SD. *P<0.05. HPV, human papilloma virus; HNSCC, head and neck squamous cell carcinoma; HB-EGF, heparin-binding epidermal growth factor; γ-H2AX, γ H2A histone family member X.