Fig. 4: PRSS35 inhibits HCC development by suppressing CXCL2-mediated neutrophil NETs formation.

a–e Hepa1-6 cells stably expressing EV, mCXCL2, Flag-mPRSS35, or mCXCL2 plus Flag-mPRSS35 were injected subcutaneously into C57BL/6J mice. a Tumor growth was measured starting from 15 days after inoculation (n = 5 biological replicates). b Western blot analysis of mCXCL2, Flag-mPRSS5 and H3Cit protein levels in tumor lysates. Ponceau staining and β-actin served as a loading control. c Tumors were extracted at the end of the experiment. The frequency of neutrophil in tumors was detected by Flow cytometry (n = 5 biological replicates). d Serum NETs levels were measured by MPO–DNA ELISA kit. e Representative IHC images (left panel) and quantification (right panel) of neutrophil in the tumor (n = 5 biological replicates). f, g Plasmids expressing YAP-5SA alone or YAP-5SA plus mCXCL2 shRNAs together with plasmids expressing PB transposase were delivered into mPRSS35-KO or WT C57BL/6J mice by hydrodynamic injection. YAP-5SA induced liver tumorigenesis was analyzed approximately 100 days after injection. f Liver/body weight ratios were measured at the end of the experiment (n = 5 biological replicates). g The frequency of neutrophil in livers was detected by Flow cytometry (n = 5 biological replicates). h–k Hepa1-6 cells stably expressing EV or Flag-mPRSS35 were injected subcutaneously into C57BL/6J mice, followed by intratumor and peritumoral injection of Ly6G antibody or IgG antibody nine days later. h Tumor growth was measured starting from 15 days after inoculation (n = 5 biological replicates). i Tumors were extracted at the end of the experiment. The frequency of neutrophil in tumors was detected by Flow cytometry (n = 5 biological replicates). j Western blot analysis of mCXCL2, Flag-tag and H3Cit proteins in tumor lysates. Ponceau staining and β-actin served as a loading control. k Serum NETs levels were measured by MPO–DNA ELISA kit (n = 5 biological replicates). Data are presented as the mean ± s.d. (a, c–i, k). Statistical significance was determined by two-way ANOVA (a, c–i, k). The blotting experiments were repeated at least three times with biological replicates (b, j). Source data are provided as a Source Data file.