## 2 Materials and Methods
### Inhibition of ARG1-Polyamine Axis Leads to Accelerated HSC Activation  
In order to investigate the impact of ARG1 activity on HSC quiescence and activation, we chose three inhibitors of the L-arginine metabolism illustrated in Figure 3A. The NO synthase inhibitor N\({}^{5}\)-(1-iminoethyl)-L-ornithine (L-NIO) inhibits the conversion of L-arginine to L-citrulline and NO. N\({}^{\omega}\)-hydroxy-L-arginine (nor-NOHA) inhibits the activity of arginase and DL-\(\alpha\)-difluoromethylornithine (DFMO) blocks the ornithine decarboxylase I (ODC1), which is the first and rate-limiting enzyme in the polyamine synthesis that converts L-ornithine to putrescine. HSCs were cultured with 0.5 mM L-NIO or nor-NOHA or 1 mM DFMO final concentrations for 4 days. Cell culture supernatant was taken from each condition every  
Figure 3: Impact of L-arginine metabolism manipulation on HSC activation. (**A**) Overview of L-arginine metabolism by ARG1 and iNOS and further conversion of L-ornithine to polyamines. Inhibitors of the pathway (iNOS: L-NIO; Arginase: nor-NOHA; ODC1: DFMO) are illustrated in red. (**B**) Urea production of HSCs day 1–4 via colorimetric urea assay in the cell culture supernatants relative to medium control. Significant differences were detected for: untreated—nor-NOHA d1: \(p\) = 0.00315; d2: \(p\) = 0.00517; d3: no sig. ; d4: \(p\) = 0.00192/ untreated—L-NIO d1: no. sig. ; d2: no sig. ; d3: \(p\) = 0.00316; d4: \(p\) = 0.000934/ untreated—DFMO d1-4: no sig. (n = 3) (**C**) Cell viability of HSCs day 0–8 with and without inhibitor treatment via Cell titer blue assay relative to blank medium. The cell viability is correlating proportional with the cell number. (n = 3) (**D**) HSCs at day 1–4 in untreated, nor-NOHA, L-NIO, or DFMO treated conditions and stained with Oil Red O to highlight lipid droplets. The scale bar indicates 0.025 mm. (**E**) Quantitative analysis of ORO-stained droplet number and area (see **D**) of n = 5 cells for each day and condition. Pictures were analyzed by ImageJ and plotted via Prism. The difference between untreated control and nor-NOHA treatment at day 3 was significant (\(p\) = 0.0308). All statistics were obtained via multiple comparison unpaired \(t\)-test using Prism 6. The error bars indicate S.D. day without medium change to determine urea production via a colorimetric urea assay (n = 3). The urea measurement showed a strong increase of urea production and release in the supernatant at day 1 of untreated cells. The urea levels on day 2, 3, and 4 were quite steady. The inhibitory effect of nor-NOHA on ARG1 activity and therefore urea production could be confirmed by almost no detectable urea concentration at day 1 and significantly reduced urea production at day 2 and 4. Interestingly, the NO synthase inhibitor L-NIO caused higher urea levels in the cells compared to all other conditions, most likely due to more substrate availability for arginase. DFMO treatment had no significant effect on the urea level compared to the untreated control (Figure 3B). To confirm that the increased or decreased urea concentrations were not based on a shift in total cell number, a cell viability assay was performed from day 0 to 8 (n = 3). The total cell number did not change up to day 3. At day 4 the cell number increased for untreated, nor-NOHA and L-NIO treated cells, but not DFMO-treated cells. Over the period of 8 days, DFMO-treated cells displayed a considerably lower cell proliferation rate compared to the other conditions, most likely caused by the reduced polyamine concentration in the cells. The addition of nor-NOHA did not change the cell viability and the corresponding cell number (Figure 4C). To detect the activation state of the HSCs under the same conditions used earlier, one 24-well was used each day for Oil Red O (ORO) staining, which stains and highlights the lipid droplets within the cytoplasm that get smaller and reduced in number during HSC activation. The ORO staining of the HSCs displayed many big lipid droplets at day 1 for all conditions.