Fig 2: Association with IP3R1 of DISC1 in cooperation with EXOC1.(a) Enhanced ER localization of Flag-DISC1 upon EXOC1 overexpression in HEK293 cells. Statistical quantification of ER localization of Flag-DISC1 was performed with densitometric analysis using Image J. DISC1 level was normalized to calnexin (n = 3). (b) Reduced ER localization of Flag-DISC1 upon EXOC1 knockdown in HEK293 cells. Statistical quantification of ER localization of Flag-DISC1 was performed with densitometric analysis using Image J. DISC1 level was normalized to calnexin (n = 3). (c) Co-immunoprecipitation of Flag-DISC1 with endogenous IP3R1 in HEK293 cells. (d) Co-immunoprecipitation of three DISC1 fragments (residues 1–350, 345–591, 586–854) with endogenous IP3R1. Statistical quantification of the IP3R1 binding levels of DISC1 was carried out via densitometric analysis using Image J (n = 3). (e) Reduced IP3R1 interaction with DISC1 upon EXOC1 knockdown in HEK293 cells. Statistical quantification of the IP3R1 binding levels of DISC1 was carried out via densitometric analysis using Image J. DISC1 level in IP lane was normalized to input lane (n = 6). (f) Reduced PKA-mediated phosphorylation of IP3R1 in DISC1 knockdown HEK293 cells (i) and mouse neuroblastoma CAD cells (ii) (n = 6 for i, 7 for ii). Error bars represent means ± SEM. *; P < 0.05, **; P < 0.01, ***; P < 0.001 (two-tailed t-test). Western blotting was performed under the same experimental conditions in each panel and full-length blots are presented in Supplementary Fig 9.

Fig 3: Apoptotic cell stimulation activates the PLC?1-IP3R axis (A,B) BMDMs were incubated with apoptotic thymocytes or live thymocytes for 10 min and lysed. Phosphor-PLC?1 (A) and phosphor-IP3R (B) in the lysates were detected by immunoblotting and quantified. ß-actin was used as a loading control. The images are representative of three independent experiments. Mean ± SEM (two-tailed unpaired Student’s t test). (C,D) BMDMs derived from Mertk-/- and WT mice were incubated with apoptotic cells for 10 min and lysed. Phospho-PLC?1 (C) and phosphor-IP3R (D) in the lysates were detected by immunoblotting and quantified. The images are representative of three (C) or four (D) independent experiments. Mean ± SEM (two-tailed unpaired Student’s t test).

Fig 4: IL-6/STAT3 inhibitor attenuated LPS-induced mitochondria-associated endoplasmic reticulum (ER) membranes (MAMs) formation, intracellular Ca2+ levels, and expression of calcium signaling-related proteins in H9c2 cells. For LPS stimuli, H9c2 cells were treated with LPS (1 µg/ml) for 12 h. For BAZ group, H9c2 cells were pretreated with 5 µM Bazedoxifene for 1 h and stimulated by LPS (1 µg/ml) for another 12 h. H9c2 cells were pretreated with 5 µM Stattic for 2 h and then the culture medium was replaced with fresh medium containing LPS for another 12 h as the Stattic group. (A–F) The levels of MAM-associated proteins FUNDC1 (B,E) and p-IP3R (Ser 1756) (C,F) in H9c2 cells and mice heart tissues were determined by western blot analysis. (G) Contact between the ER and mitochondria (Mito) was evaluated by confocal microscopy. Representative images were shown. Scale bar: 10 µm. (H) Pearson's coefficient was quantified for detecting the association between ER and mitochondria. (I) Fluo-4 staining was performed to show intracellular Ca2+ levels. Scale bar: 50 µm. (J) Quantification of Fluo-4 fluorescence intensity. (K,L) The levels of calcium signaling-related proteins Cav1.2 and RyR2 in H9c2 cells (K) and mice heart tissues (L) were determined by western blot analysis. n = 3 (A–F,K,L), and 5 (G–J) per group. Data represent means ± S.E.M. *p < 0.05 vs. control; #p < 0.05 vs. LPS treatment. Statistical analyses were performed using one-way ANOVA followed by Bonferroni's post-hoc test. BAZ, Bazedoxifene.

Fig 5: FUNDC1-knockdown prevented LPS-induced calcium signaling-related proteins expression, MAMs formation, increases of intracellular Ca2+ levels, mitochondrial dysfunction, and ROS production. H9c2 cells were transfected with control siRNA or FUNDC1 siRNA (siFUNDC1), and then treated with LPS (1 µg/ml for 12 h. (A–E) FUNDC1 (B), p-IP3R (Ser 1756) (C), IP3R, RyR2 (D), and Cav1.2 (E) levels were examined by western blot. (F,G) Representative confocal images (F) and quantification (G) of ER-mitochondria contacts. Scale bar: 10 µm. (H,I) Representative images (H) and fluorescence intensity quantification (I) of Fluo-4 staining. Scale bar: 100 µm. (J) ATP content in each group. (K) Representative images of JC-1 staining. Scale bar: 50 µm. (L) Representative images of H2DCFDA staining. Scale bar: 100 µm. (M) Quantification of the JC-1 fluorescence ratio (JC-1 aggregate/JC-1 monomer). (N) Quantification of ROS production as measured by H2DCFDA fluorescence intensity. n = 3 (A–E,J) and 5 (F–I,K–N) per group. Data represent means ± S.E.M. *p < 0.05 vs. control; #p < 0.05 vs. LPS treatment. Statistical analyses were performed using one-way ANOVA followed by Bonferroni's post-hoc test. BAZ, Bazedoxifene.