Fig. 5
NETs internalized by HCC cells upregulated COX2 through activation of TLR4/9. a Representative fluorescent images of NET internalization by HepG2 and MHCC97H cells (left panel) and flow cytometry analysis (right panel). NETs were labeled with SytoxGreen and incubated with HepG2 and MHCC97H cells for 6 h. The HCC cells were then fixed, stained for nuclear, and observed under fluorescence microscope or analyzed by flow cytometry. The white triangle indicated NETs internalized. Scar bar: 20 μm. b RT-PCR screening of mRNA level changes in chosen DAMP sensors in HepG2 and MHCC97H cells treated with NETs. Results were presented as log2 fold change relative to untreated cells. c Western blot analysis of COX2, TLR4/9, and P-P65 levels in HepG2 and MHCC97H cells treated with NETs. Cells were transfected with corresponding siRNA and stimulated with NETs for 6 h. d, e The cytotoxicity (d) and invasion capacity alteration (e) on siRNA transferred HepG2/MHCC97H cells followed by NET treatment and PGE2 rescue. *, #: P < 0.05; **,##: P < 0.01. Data were presented as means ± SEM