Fig. 4: Macropinocytosis of CPPs depends on CaSR signaling.
a [Ca2+]-dependent uptake of calcein-stained CPPs (calcein-CPP) was visualized and analyzed with ImageStreamX Mark II. 5,000–10,000 freshly isolated human blood monocytes from eight donors were imaged and analyzed for calcein fluorescence after 45 min incubation with calcein-CPPs in 1 mM [Pi] RPMI1640/10%FBS. Representative images are shown for monocytes incubated with calcein-CPPs in the absence (left) and presence (right) of 2.5 mM added [Ca2+]. b Uptake of calcein-stained CPPs (calcein-CPP) after incubation with the indicated added concentrations of [Ca2+], or [Ba2+] in RPMI1640/10%FBS containing 1 mM [Pi] for 45 min (four donors). c Calcein-CPP uptake was analyzed in control and CaSR-deficient THP-1 cells (five experiments). Cells were incubated with calcein-stained CPPs and stimulated with 2.5 mM added [Ca2+] for 10, 20, and 30 min, as specified. d Dynamic mass redistribution (DMR) measurement in freshly isolated blood monocytes is shown as mean ± s.e.m. (dotted curves) of six experiments from six donors. Addition of agents of interest is indicated as vertical dotted line. Cells were either stimulated in 1 or 5.6 mM [Pi]-containing RPMI1640/10%FBS medium as indicated. Inset shows median of time point 10 min. e, f DMR analysis of human monocytes stimulated with 2.5 mM added [Ca2+] in RPMI1640/10%FBS medium containing [Pi] as indicated (e, two donors) and after pre-incubation with 5 µM of the Gαq inhibitor YM254890 or the corresponding DMSO dilution (control) for 60 min (f, three donors). Data are shown as mean. g Cathepsin B activity determined by magic red fluorescence in monocytes from the indicated numbers of donors stimulated with increasing [Ca2+] in RPMI1640/10% FBS containing 1 or 5.6 mM [Pi] is shown as x-fold over control (LPS); Box-and-Whisker plots show median, 25th–75th percentile, and min/max whiskers, bar charts show mean ± s.e.m., unless indicated otherwise. Statistical analysis was performed using two-tailed Mann–Whitney U test (a, c, d) or two-tailed t-test b. p-values are indicated as *p < 0.05, **p < 0.01, ***p < 0.001.
