## 4 Discussion
This hypothesis needs to be verified experimentally in future studies. The interaction of ERas and ARG1 did not show any effects on ARG1 enzymatic activity in our experimental set-up. Therefore, the interaction of ERas and ARG1 may have other functional significance that can only be speculated about at this point. On the one hand, ARG1 and iNOS, which are reciprocally expressed during HSC activation, might each directly control the other's expression. The elevated expression of iNOS, one day after isolation and cultivation outside of their stem cell niche, is likely to be a stress response, driven by the transcription factor NFkB that regulates iNOS mRNA expression [45]. NFkB was furthermore described to play an essential role in both profibrogenic and antifibrogenic signaling pathways during liver diseases, and was also investigated in HSCs [46; 47]. The expression level of iNOS is not only controlled by NFkB, but also by the translation initiation factor 2\(\alpha\) (eIF2\(\alpha\)), which is in turn regulated by L-arginine levels and therefore likely to be connected to ARG1 activity, also shown in astrocytes which have multiple similarities with HSCs [48; 49; 50]. On the other hand, the interaction of ERas and ARG1 could recruit ARG1 to a specific subcellular localization on lipid membranes to either increase ARG1 activity as it was shown in red blood cells, where the activity of arginase was approximately 100 times higher in membrane fractions compared to cytoplasmic fractions [51], or to source exogenous but not endogenous L-Arg, according to observations in other cells types, thereby solving \"the arginine paradox\" [52]. ERas-ARG1 interaction might recruit ARG1 to the plasma membrane close to a cationic amino acid transporters (CATs) whose paralogs considerably differ in their affinity for L-arginine (CAT1: 0.10-0.16 mM; CAT2A 2-5 mM; CAT2B and CAT3: 0.25-0.70 mM) [53]. One hint might be the coexpression of ARG1 and CAT2A in quiescent HSCs that have a comparable low affinity for L-arginine and would match consumption and transportation affinity of L-arginine. For comparison, iNOS is coexpressed with the inducible CAT2B paralog in HSCs at day 1, which has also been reported for astrocytes [49; 54]. In order to analyze the formation of an ARG1-ERas-CAT2A colocalization on the plasma membrane, future extensive studies and validated antibodies, which we do not currently have, are needed. This study could successfully add arginase 1 to the list of interaction partners of ERas and to the proteins that are needed for the maintenance of quiescent HSCs. The alteration of the L-arginine metabolism _via_ arginase and ODC1 inhibitors shows a strong picture towards an accelerated activation, whereas the iNOS inhibitor slows down the development of HSCs into myofibroblast-like cells. As HSCs are located in a tightly controlled stem cell niche in the space of Disse [10], we always have to consider the effect of amino acid/polyamine depletion and delivery, as well as the communication of HSCs as liver pericytes with surrounding cells and the vascular system. The following are available online at [[https://www.mdpi.com/article/10.3390/cells11030508/s1](https://www.mdpi.com/article/10.3390/cells11030508/s1)]([https://www.mdpi.com/article/10.3390/cells11030508/s1](https://www.mdpi.com/article/10.3390/cells11030508/s1)), Figure S1: Immunoprecipitation analysis of ARG1 and _rn_ERas, Figure S2: Confocal images of ERas, ARG1 and GFAP in quiescent HSCs., Figure S3: Confocal images of ERas--ARG1 and ARG1--Na\({}^{+}\)/K\({}^{+}\)-ATPase in quiescent HSCs., Figure S4: Pull-down experiments of ERas _hs_FL, _hs_AN and _rn_/_hs_ G-domain with purified ARG1., Figure S5: Phase contrast microscopy of HSCs day 4., Figure S6: Oil Red O staining of HSCs day 1 to 4 with combination treatment of L-NIO and nor-NOHA., Figure S7. Urea assay with ARG1 and human ERas FL or Nex., Figure S8.