Figure 6. Ca²⁺ influx is provoked during mutated NLRP3-mediated inflammasome assembly.

(A–C) Differentiated TRE-NLRP3-L353P-THP-1 cells were loaded with 4 µM Fluo-8 for 1 hr and treated with doxycycline (DOX) (30 ng/mL) at 37°C for 6 hr. The images were recorded by confocal microscopy at 5 min intervals from 3 hr to 6 hr. (A) Representative temporal subtraction images. (B) The frequency of intracellular Ca²⁺ increase at each time point. (C) The cumulative number of Fluo-8-positive cells. (D–F) Differentiated TRE-NLRP3-L353P-THP-1 cells were loaded with 4 µM Fluo-8 for 1 hr and treated with DOX (30 ng/mL) at 37°C for 6 hr in Ca²⁺-depleted or -supplemented media. The images were recorded by confocal microscopy at 5 min intervals from 3 hr to 6 hr. (D) Representative temporal subtraction images. (E) The frequency of intracellular Ca²⁺ increase at each time point. (F) The cumulative number of Fluo-8-positive cells. (G–I) Differentiated EF1-ASC-BFP/TRE-NLRP3-L353P-THP-1 cells were loaded with 4 µM Fluo-8 for 1 hr and treated with DOX (30 ng/mL) at 37°C. The images were recorded at 5 min intervals. (G) Representative images of the cells with increased Fluo-8 signals. (H) The apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC)-BFP speck size (I) and fluorescent intensity of Fluo-8 were analyzed. The peak time point of Fluo-8 signals was defined as 0 min. (H) The blue line and the (I) green line represent mean values, and the gray line represents each measurement. *p<0.05, **p<0.01, and ***p<0.005 as determined by (C and F) Fisher’s exact test with the Holm correction or (H and I) repeated one-way ANOVA with a post hoc test. (A–G) Data are representative of three independent experiments. (H and I) Data are from three independent live-cell imaging.

Figure 6—figure supplement 1. Ca²⁺ influx is induced by chronic infantile neurological, cutaneous, and articular syndrome-associated NLRP3 mutant.

(A–C) Differentiated TRE-NLRP3-D303N-THP-1 cells were loaded with 4 µM Fluo-8 for 1 hr and treated with doxycycline (DOX) (30 ng/mL) at 37°C for 6 hr. The images were recorded by confocal microscopy at 5 min intervals from 3 hr to 6 hr. (A) Representative temporal subtraction images. (B) The frequency of intracellular Ca²⁺ increase at each time point. (C) The cumulative number of Fluo-8-positive cells. ***p<0.005 as determined by Fisher’s exact test with the Holm correction. Data are representative of three independent experiments.

Figure 6—figure supplement 2. Pyroptosis is induced by mutated NLRP3 after Ca²⁺ influx.

(A and B) Differentiated TRE-NLRP3-L353P-THP-1 cells were loaded with 4 µM Fluo-8 for 1 hr and treated with doxycycline (DOX) (30 ng/mL) at 37°C for 6 hr. (A) Representative images of cells with increased Fluo-8 signal. (B) The relative fluorescent intensity of Fluo-8 and Kusabira Orange in cells with increased Fluo-8 signal was quantified (n=10). (C and D) Differentiated TRE-NLRP3-L353P-THP-1 cells were loaded with 4 µM Fluo-8 for 1 hr and treated with DOX (30 ng/mL) at 37°C for 6 hr in the presence of SYTOX Deep Red. (C) Representative images of cells with increased Fluo-8 signal. (D) The relative fluorescent intensity of Fluo-8 and SYTOX Deep Red in cells with increased Fluo-8 signal was quantified (n=5). Data are expressed as the mean ± SD. *p<0.05 as determined by repeated one-way ANOVA with a post hoc test. Data are analyzed from two independent time-lapse imaging.