Fig. 6: Calciprotein particles are not degraded in the lysosomes.

a Quantification of cell elongation of calcium-differentiated CaSR-deficient THP-1 cells in the presence (PMA) or absence (control) of PMA. A total of 200 cells per condition from 4 different experiments were analyzed. Bar chart show mean ± s.e.m. Statistical analysis was performed using two-tailed t test. a–d Calcium-dependent uptake of calcein-labeled CPPs (b) and co-localization of calcein-CPPs with lysosomes (c) stained with lysotracker in monocytes stimulated with and without 2.5 mM CaCl₂ for 4 h. Analysis was performed using ImageStreamX Mark II and 5000–10,000 monocytes were imaged from 3 different donors. Representative images showing brightfield (BF), calcein-CPPs, lysotracker, and an overlay of CPP and lysotracker staining (d). Bar charts show mean ± s.e.m. Statistical analysis was performed using two-tailed t test. e-g Uptake of DQ-BSA in calcein-CPP + and CPP- negative monocytes (e) and co-localization of calcein-CPPs with DQ-BSA in calcein-CPP + monocytes (f) stimulated with 2.5 mM CaCl₂ for 4 h. Analysis was performed using ImageStreamX Mark II and 5000–10,000 monocytes were imaged from 3 different donors. Representative images showing calcein-CPPs, DQ-BSA, and an overlay of CPP and DQ-BSA staining (g). Bar chart show mean ± s.e.m. Statistical analysis was performed using two-tailed t test. h–j Analysis of lysosomes using lysotracker in monocytes stimulated with and without 2.5 mM CaCl₂ for 3 h. Analysis was performed using ImageStreamX Mark II and 5000–10,000 monocytes were imaged from 4 different donors. Bar chart show mean of median fluorescence ± s.e.m. Statistical analysis was performed using two-tailed t test (h). Representative histogram of lysotracker fluorescence in control monocytes (white) and calcium-treated monocytes (red) (i), and representative images showing brightfield (BF) and lysotracker (LT) (j). k–m Analysis of lysosomes using LAMP2 in monocytes stimulated with and without 2.5 mM CaCl₂ for 3 h. Analysis was performed using ImageStreamX Mark II and 5000–10,000 monocytes were imaged from 4 different donors. Bar chart show mean of median fluorescence ± s.e.m. Statistical analysis was performed using two-tailed t test (k). Representative histogram of LAMP2 fluorescence in control monocytes (white) and calcium-treated monocytes (red) (l), and representative images showing brightfield (BF) and LAMP2 (m). n, o LysoSensor staining of differentiated control THP-1 cells (CaSR wt) or CaSR-deficient THP-1 cells (CaSR ko) incubated in the presence (Ca²⁺) or absence (con) of calcium for 3 h. Analysis was performed using ImageStreamX Mark II and 5000–10,000 monocytes were imaged from 3 different experiments. Representative images showing brightfield (BF) and LysoSensor (LS) (n). Violin plot show median of the mean intensity of lysosomes. A total of 168 cells and 6900–8500 lysosomes were analyzed per condition. Statistical analysis was performed using Mann–Whitney U-test (o). p Violin plot of bovine Fetuin-A abundance in the proteome of calcium-macrophages (n = 11), GM-CSF-macrophages (n = 5), and M-CSF-macrophages (n = 5).