Fig. 1: In the presence of [Pi] and fetuin-A, addition of [Ca2+] triggers calciprotein particle formation. | Nature Communications

Fig. 1: In the presence of [Pi] and fetuin-A, addition of [Ca2+] triggers calciprotein particle formation.

From: Calcium-sensing receptor-mediated NLRP3 inflammasome response to calciprotein particles drives inflammation in rheumatoid arthritis

Fig. 1

a Differentiated CaSR-deficient (def-CaSR) or control THP-1 cells incubated with LPS and either the indicated amount of [Ca2+], 3 mM ATP, or 100 µg/ml MSU in RPMI1640/10%FBS containing 5.6 mM [Pi] for 8 h (numbers of experiments as indicated). b Blood monocytes of myeloid CaSR-KO mice (B6.129P2-Lyz2tm1(cre)Ifo7jxCaSR∆flox/∆flox, n = 9) or control mice (B6.129P2-Lyz2tm1(cre)Ifo7jxCaSRWT/WT, n = 8) were treated for 16 h with LPS (ctrl), LPS plus 2.5 mM added [Ca2+] or LPS plus 100 µg/ml MSU in RPMI1640/10%FBS containing 5.6 mM [Pi]. Blood from 2 to 3 mice was pooled, and used in three experiments. c Freshly isolated human blood monocytes from the indicated numbers of donors were incubated with LPS and the indicated amount of [Ca2+] in RPMI1640/10%FBS containing the indicated [Pi] for 16 h. a–c IL-1β was detected in supernatants. Box-and-Whisker plots show median, 25–75th percentile, and min/max Whiskers.Wilcoxon signed-rank test a or two-tailed Mann–Whitney U test b, c was used. p-values are indicated as *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 (or # respectively). #Indicates level of significance for comparison to control (LPS) in b and for comparison between 1.5 and 2.5 mM added [Ca2+] in c; *Indicates level of significance for comparison between 0 and 2.5 mM added [Ca2+] in c. d, e Dynamic light scattering analysis (main peak intensity) of nanoparticles forming in RPMI1640/10%FBS. d Particle size (two experiments with technical triplicates) and [Ca2+] (one experiment) in relation to [Pi] after addition of 2.5 mM [Ca2+]. Data show mean. e Time course of particle size and [Ca2+] after addition of 2.5 mM CaCl2 to RPMI1640/10%FBS with 5.6 mM [Pi]. f Western Blot detecting fetuin-A in isolated calciprotein particles (CPPs). Shown is one representative experiment out of three. g, h Transmission electron microscopy (TEM) and Cryo-TEM imaging (n = 2) of particles formed spontaneously after 2 h in RPMI1640/10%FBS and 2.5 mM [Ca2+]. i Scanning transmission electron microscopy (STEM) high angle annular dark field (HAADF) image with energy-dispersive X-ray spectroscopy (EDX) signals for Ca (blue) and P (yellow) of particles present after 2 h of incubation in RPMI1640/10%FBS and 2.5 mM [Ca2+] (n = 2). j EDX-spectrum of particles shown in i.

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