Supplementary Material for: Endocytotic Albumin Uptake Pathways in Human Adipose Stem Cells and Connection to Intracellular Calcium Oscillations and the Neonatal Fc receptor (FcRn)

Intracellular Ca2+ oscillations of unknown function occur in human adipose tissue stem cells (ASCs). In the present study we investigated whether Ca2+ oscillations in ASCs from subcutaneous fat tissue derived from female patients were driving albumin endocytosis by involving the neonatal Fc receptor (FcRn), which is mediating the recycling of albumin and IgG. Our data demonstrated that endocytosis of albumin occurred by micropinocytosis (inhibition by cytochalasin D, wortmannin, and ethylisopropylamiloride (EIPA)), caveolae-dependent (inhibition by genistein and nystatin) and clathrin-mediated (inhibition by Dyngo-4a) pathways. In serum-free medium Ca2+ oscillations were absent, but could be induced by addition of either fetal bovine serum (FBS), albumin, IgG or a stimulating antibody against FcRn. Consequently, FcRn expression was demonstrated in ASCs by western blot analysis and immunohistochemistry, and colocalized with fluorescence-labelled, endocytosed albumin. Ca2+ oscillations were inhibited by the Ca2+ chelating agent BAPTA, the store-operated Ca2+ entry (SOCE) inhibitor SKF96365, the Ca2+ sensing receptor (CaSR) inhibitor NPS-2143, the macropinocytosis inhibitors cytochalasin D, wortmannin, and EIPA, the caveolae-dependent endocytosis inhibitors genistein and nystatin, and the clathrin-mediated endocytosis inhibitor Dyngo-4a. Uptake of fluorescence-labelled albumin was inhibited by agents interfering with Ca2+ oscillations and endocytosis blockers. Notably, not only intracellular albumin and IgG accumulation, but also Ca2+ oscillations were inhibited by the FcRn-blocking antibody nipocalimab which interferes with the IgG binding site of FcRn. Moreover, siRNA-mediated downregulation of FcRn protein expression significantly reduced intracellular albumin content, the number of cells displaying Ca2+ oscillations, and the duration and amplitude of Ca2+ signals. In summary, our data suggest that Ca2+ oscillations in human ASCs regulate albumin uptake and presumably IgG recycling via FcRn.