Fig. 5
PADI4 in macrophages mediated MET formation triggered by sorafenib/IL4. A Detection of protein expressions of molecules associated with MET formation in M2-mBMDM using Western Blot. NC-CM: media of Hepa1-6. Sora-CM: media of Hepa1-6 treated by sorafenib. B The mRNA expression of PADI4 in M2-mBMDM and M2-THP-1. NC: M2-mBMDM and M2-THP-1 received treatment from media of Hepa1-6 or Hep3B cells. Sora-CM: M2-mBMDM and M2-THP-1 received treatment from media of Hepa1-6 or Hep3B cells that were treated by sorafenib. n = 3. C The Western Blot assay of PADI4 protein expression in M2-mBMDM and M2-THP-1 cells that were treated with HCC cell media. NC-CM: media of Hepa1-6 or Hep3B. NC-CM + IL4-IN: received treatment with IL4 inhibitor (IL4-IN, neutralizing antibody) and NC-CM. Sora-CM: media of Hepa1-6 or Hep3B treated by sorafenib. Sora-CM + IL4-IN: received treatment with IL4-IN and Sora-CM. ELISA detection of the D MPO-DNA and G elastase concentration in the supernatant of M2-mBMDM and M2-THP-1 cells that were treated with media of HCC cell Hepa1-6 or Hep3B. E Microscopy detection of SYTOX Green fluorescence in M2-THP-1 cells that were treated with media of Hep3B cells. Scale bar: 400 μm. F The intensity of SYTOX Green fluorescence in M2-mBMDM and M2-THP-1 cells that were treated with media of HCC cell Hepa1-6 or Hep3B. NC: media of HCC cells. Sora: media of HCC cells receiving treatment with sorafenib. Sora + IL4-IN: received treatment with IL4-IN and media of HCC cells treated by sorafenib. Sora + PADI4-IN: received treatment with PADI4 inhibitor (PADI4-IN, GSK484) and media of HCC cells treated by sorafenib. H Tumor growth curve in Hepa1-6 model of HCC. I Tumor volume in Hepa1-6 model of HCC on day 25 after the first injection. J Survival of Hepa1-6 model of HCC. NC: negative control with saline treatment. Sora: treatment with sorafenib. PADI4-IN: treatment with PADI4 inhibitor GSK484. Sora + PADI4-IN: treatment with sorafenib and GSK484. n = 6 (D, F, G, I and J). Statistical significance was calculated by two-tailed student’s t-test (B), one-way analysis of variance (ANOVA) followed by Tukey’s multiple comparisons test (D, F, G and I), and Log-rank (Mantel-Cox) test (J). Data represent the mean ± standard deviation (SD) of ≥ three independent experiments. *, p < 0.05; **, p < 0.01; ***, p < 0.001