*3.2. S100A6 Represents a Marker for Activated Myofibroblasts*

At present, α-SMA is largely accepted as a marker of activated MFB [12]. However, in our scRNASeq analysis, this marker only recognized a subset of activated MFB (see Figure 1C). We therefore wanted to identify markers that are uniquely and uniformly upregulated on all subsets of activated MFB in vivo (Figure 2A). We found that the S100 calcium binding protein A 6 (*S100a6*) is highly upregulated on activated MFB but not on resting HSCs (Figure 2A,B). We first confirmed the presence of S100A6 positive cells in the periportal (i.e., fibrotic) areas of CCl4-treated livers by immunohistochemistry (Figure 2C). By using immunofluorescence co-staining for PDGFR-β (red) and S100A6 (green) we could then confirm the presence of double positive cells, corroborating the MFB phenotypes observed by our scRNASeq analysis (Figure 2D).

**Figure 2.** The S100 calcium binding protein A 6 (S100A6) expression marks activated myofibroblasts. (**A**) Differential gene expression analysis of HSCs versus MFB, showing the top five genes upregulated in both groups. (**B**) t-SNE plot of relative gene expression of S100A6, based on scRNASeq analyses from normal and fibrotic mouse livers. (**C**) Representative image of immunohistochemistry staining for S100A6 in CCl4-treated fibrotic liver. (**D**) Representative images of immunofluorescence co-staining for PDGFR-β (red) and S100A6 (green) on untreated and CCl4-treated fibrotic liver. Nuclei are stained with DAPI (4 ,6-diamidino-2-phenylindole, blue). Scale bar represents 100 μm.
