*3.3. Di*ff*erential Expression of Chemokines and Collagens by Activated Myofibroblast Subsets*

Upregulated expression of chemokines, such as CCL2, CXCL1, or CXCL12, and collagens, particularly COL1A2, COL3A1, or COL5A2, are often named attributes for activated MFB [13]. We herein wanted to analyze whether different subsets of either chemokine or collagen-producing MFB sub-clusters exist in fibrotic livers in vivo (Figure 3A). By scRNASeq analysis, the expression of collagens is homogenously upregulated in activated MFB, while chemokines show a more restricted pattern. While the monocyte recruiting chemokine *Ccl2* is expressed by both resting HSCs and MFB, neutrophil recruiting *Cxcl1* is strongly associated with activated MFB only. *Cxcl12,* on the other hand, is highly expressed by both HSCs and MFB, except for cluster MFB II, representing myeloid myofibroblasts (Figure 3A,B and Supplementary Table S1). By correlating the expression of the chemokine *Ccl2* with *Col3a1* on a single-cell level, resting HSCs comprised cells that express only *Col3a1*, cells co-expressing *Col3a1*, or cells that only express *Ccl2*. Interestingly, activated MFB could be differentiated by their expression of *Ccl2*, while all cells highly expressed *Col3a1* (Figure 3C). These data confirm the universal importance of an upregulated collagen expression during the activation of HSCs to MFB, while resting HSCs and selected MFB clusters demonstrate the capacity to secrete chemokines and thereby modulate the inflammatory environment in their surroundings.

**Figure 3.** Differential chemokine and collagen gene expression patterns in hepatic stellate cells and myofibroblasts. (**A**) Feature plots showing the relative gene expression strength of selected marker genes. (**B**) Violin plots showing the relative gene expression of activation markers. (**C**) Gene plots for the normalized gene expression of *Col3a1* and *Ccl2*. *n* = 4 with an average of 5000 cells per condition and ~60,000 reads per cell.
