Figure 4.

HCC cells-derived CXCL8/IL-8 triggers TAN towards NETs formation through NADPH pathway. (A) The protein expression of IL-8 was detected by Western blot assay in three HCC cell lines. Experiment was repeated three times. (B) The expression of IL-8 in HCC tissues was examined by the immunohistochemistry (n=30). A representative image was shown. Scare bar: 100um. (C) Co-incubation of HD neutrophils (2.5x10⁶/mL) with the conditioned medium of HCC cells (HCC-CM) (1:2) was performed to explore the effect of IL-8 on HCC-CM-induced NETs formation. The expression of CitH3 was detected by immunofluorescence staining on coverslip for the identification of NETs formation. Representative images were shown. IL-8 neutralizing antibody was added into the culture medium at the concentration of 5µg/mL. NOX inhibitor apocynin was added into medium at the final concentration of 10uM. Experiment was repeated three times. Representative images were shown. The white arrows point to the presence of NETs-CitH3. Scare bar: 100µm.