Fig. 4: Effects of apoptotic hepatocytes and rMFG-E8 on inflammatory gene expression in MFG-E8 KO macrophages.

a Immunoblot analysis of MFG-E8 and ERK (loading control) in parental wild-type (WT) Hepa1c1c7 cells and Hepa1c1c7 cells in which endogenous Mfge8 was deleted using the CRISPR–Cas9 system. Cells were exposed to staurosporine to induce apoptosis (three replicates shown), a known stimulus for MFG-E8 expression. b RT–qPCR analysis of Tnf, Ccl2, Il1b, and Il6 mRNA abundance in thioglycolate-elicited peritoneal macrophages (n = 4–5) cocultured directly with apoptotic Mfge8-deficient Hepa1c1c7 cells. c RT–qPCR analysis of Tnf, Ccl2, Il1b, and Il6 mRNA abundance in thioglycolate-elicited peritoneal macrophages (n = 3) cocultured with apoptotic Mfge8-deficient Hepa1c1c7 cells across a Transwell membrane. In (b, c), apoptosis was induced in Hepa1c1c7 cells by staurosporine treatment. The apoptotic hepatocytes were cocultured with macrophages for 3 h under the indicated conditions in the presence or absence of recombinant MFG-E8 protein (rMFG-E8; 1.0 µg/ml). Cells were then harvested for RT–qPCR analysis. Marker shapes and colors in the bar graphs indicate macrophage culture conditions: black circles, macrophages alone (Mfs); blue squares, macrophages cocultured with dead hepatocytes (Mfs + Hepa); red triangles, macrophages cocultured with dead hepatocytes in the presence of rMFG-E8 (Mfs + Hepa + rMFG-E8). The amounts of mRNAs were normalized to 18S rRNA. Data are means ± s.e.m. *P < 0.05, **P < 0.01 (one-way ANOVA and Tukey’s post hoc test).