*5.7.1 Gram-reaction test*

Gram reaction has been accomplished according to Gregersen [30], adopting a 3% KOH solution, wherein one colony is dissolved and then the loopful is removed and checked for viscosity. A significant viscosity change is indicative of Gram negativity, whereas an unchanged viscosity reveals the Gram positivity. The rationale is based on Gregersen's observation that Gram+ withstand potassium hydroxide whereas in Gram- cellular wall is destroyed and DNA is released resulting in a viscosity change.


**5. Conclusions**

The major goal of this research was to clarify whether or not the expression of the tricellular tight junction (tTJ) protein, angulin-1, is involved in controlling paracellular water transport in cell lines with different levels of tightness (HT-29/B6 and MDCK C7 cells). This work supports the previous hypothesis that tTJs function differentially in distinct epithelial cell lines.

In brief, we provide insights into tTJ permeability by showing that angulin-1 can function as regulator of the macromolecular barrier in the tricellular tight junction regardless of the tightness of the epithelium (Supplementary Table S3). Manipulating the tTJ permeability (via tricellulin or via angulin-1) represents a mechanism to regulate epithelial water homeostasis only in tight epithelial cell lines (MDCK C7 cells); nevertheless, in intermediate-tight epithelial cell lines (HT-29/B6 cells) the contribution of tTJ seems to be negligible. Thus, the contribution of tTJ to transepithelial water transport depends on the tightness of the epithelium. Probably, then, the regulation of water paracellular transport by osmolytes in each type of epithelial cell has physiological significance depending on the organ to which it belongs.

**Supplementary Materials:** Supplementary Materials can be found at https://www.mdpi.com/1422 -0067/22/15/7827/s1.

**Author Contributions:** Conceptualization, C.A.-T., S.M.K., R.R. and M.F.; funding acquisition, M.F.; investigation, C.A.-T.; methodology, R.R. and M.F.; supervision, S.M.K., R.R. and M.F.; validation, S.M.K., R.R. and M.F.; visualization, C.A.-T.; writing—original draft, C.A.-T.; writing—review and editing, S.M.K., R.R. and M.F. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by the Deutsche Forschungsgemeinschaft (DFG), Research Training Group GRK 2318, and the Open Access Publication Fund of the Charité—Universitätsmedizin Berlin.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Data is contained within the article or Supplementary Materials.

**Acknowledgments:** We would like to thank Britta Jebautzke, In-Fah M. Lee, and Anja Fromm (Clinical Physiology/Nutritional Medicine, Medical Department, Division of Gastroenterology, Infectiology and Rheumatology, Charité—Universitätsmedizin Berlin, 12203 Berlin, Germany) for their expert technical assistance.

**Conflicts of Interest:** The authors declare no conflict of interest.