Fig 2: 3-HB inhibits ER stress-induced activation of NLRP3 inflammasome via stabilizing ER Ca2+ storage. A,B) Western blot analysis of ER stress sensors (PERK, ATF-6, and IRE1a) changes of BMDMs’ cell lysate. BMDMs were stimulated with LPS (100 × 10-9 g mL-1) and then added with the IP3R inhibitor 2-APB (100 × 10-6 m)/IP3R agonist Ins(1,2,4,5)P4) (1 × 10-6 m) with or without 3-HB (10 × 10-3 m). C,D) 3-HB inhibits ER stress-induced NLPR3 inflammasome activation. BMDMs were stimulated with LPS (100 × 10-9 g mL-1) to mimic the microenvironment of plaque, and meanwhile co-incubated with IP3R inhibitor 2-APB (100 × 10-6 m)/ IP3R agonist Ins(1,2,4,5)P4) (1 × 10-6 m) with or without 3-HB (10 × 10-3 m), and then analysis of the mature products (IL-18 and IL-1ß) of NLRP3 inflammasome activation. The data are presented as mean±SEM from at least three independent experiments and analyzed by the one-way ANOVA, * p<0.05, ** p<0.01.

Fig 3: 3-HB inhibits Ca2+ releasing from ER depended on 3-HB–Gpr109a signaling-activated Ca2+ influx. A,B) Change in cytosolic Ca2+ concentration in real time as detected by Fura-2 fluorescence imaging. The isolated BMDMs from WT or Gpr109a -/- mice were loaded with Fura-2 AM (10 × 10-6 m), and then perfused in Hank's buffer (with added Ca2+). 3-HB (1 × 10-3, 5 × 10-3, and 10 × 10-3 m) or niacin (0.1 × 10-6 m) were added as indicated by the vertical lines in the Ca2+ tracings. C) Increases in intracellular Ca2+ concentration after the addition of 3-HB (1 × 10-3, 5 ×10-3, and 10 × 10-3 m) or niacin (0.1 × 10-6 m) were calculated and plotted (n = 5). D) Change in cytosolic Ca2+ concentration in real time detected via Fluo-4 fluorescence imaging. The isolated BMDMs from WT mice were loaded with Fluo-4 AM (10 × 10-6 m), and then perfused in Hank's buffer (without addition of Ca2+). LPS (100 × 10-9 g mL-1), LPS (100 × 10-9 g mL-1) +3-HB (10 × 10-3 m) or LPS (100 × 10-9 g mL-1) +2-APB (100 × 10-6 m) were added as indicated using Ca2+ tracking. E) Increases in intracellular Ca2+ concentration after the addition of LPS (100 × 10-9 g mL-1) +3-HB (10 × 10-3 m) or LPS (100 × 10-9 g mL-1) +2-APB (100 × 10-6 m) (n = 5). F,G) Changes of phosphorylation levels of protein PLC, IP3R, and CaMKII in BMDMs treated with LPS (100 × 10-9 g mL-1) with or without 3-HB (10 × 10-3 m) or the intracellular calcium chelater BAPTA-AM (20 × 10-6 m). Data are presented as mean±SEM from at least three independent experiments, one-way ANOVA. ** p <0.01, *** p <0.01, **** p <0.01; NS, no statistical significance.