Fig 1: Apoptotic cell stimulation induces the Orai1-STIM1 association in a PS-dependent manner (A) LR73 cells transfected with the indicated plasmids were incubated with apoptotic cells or apoptotic cell supernatants for 10 min. Orai1-FLAG in cell lysates was precipitated with anti-FLAG antibody-conjugated agarose beads. Bound proteins were detected by immunoblotting. (B,C) LR73 cells were incubated with TAMRA-stained apoptotic cells for 2 h in the presence of the indicated concentration of cytochalasin D (1 µM) (B) or bafilomycin A (1 µM) (C), and engulfing phagocytes were analyzed by flow cytometry. n = 3 experiments, mean ± SEM (two-tailed unpaired Student’s t test). (D–F) LR73 cells transfected with the indicated plasmids were stimulated with apoptotic cells for 10 min in the presence or absence of cytochalasin D (1 µM) (D), bafilomycin A (1 µM) (E), or Mfge8D89E (F). The Orai1-STIM1 association was detected as in (A). (G) LR73 cells transfected with Orai1 and STIM1 were stimulated with PC or PS liposomes for 10 min. Cell lysates were incubated with anti-FLAG antibody-conjugated agarose beads. Bound proteins were detected with the indicated antibodies. The images are representative of at least three independent experiments (A,D–G).
Fig 2: Mertk depletion attenuates the Orai1-STIM1 association and calcium entry (A) BMDMs derived from Mertk−/− and WT mice were incubated with apoptotic cells for 10 min. Cell lysates were incubated with an anti-Orai1 antibody and protein A/G-conjugated agarose beads. Bound proteins were detected with the indicated antibodies (left) and co-immunoprecipitated STIM1 with Orai1 was quantified (right). The arrow heads indicate Orai1. The images are representative of three independent experiments. Mean ± SEM (two-tailed unpaired Student’s t test). (B) BMDMs derived from Mertk−/− and WT mice were stained with Fluo4 and incubated with apoptotic cells. The MFIs of Fluo4 in the cells were analyzed by flow cytometry. n = 5 experiments, mean ± SEM (two-way ANOVA). (C–E) BMDMs from the indicated mice were stained with Fluo4 and then treated with 0.1 µM thapsigargin for the indicated duration. Thereafter, apoptotic or live thymocytes in medium containing 1.0 mM calcium were added to the cells at the indicated time. Fluorescence of the cells was measured with a microplate reader. Data are representative of 4 independent experiments (C), and the peak and slope of SOCE were calculated (D,E). n = 3 experiments, mean ± SEM (two-way ANOVA).
Fig 3: Apoptotic cell stimulation induces the Orai1-STIM1 interaction in phagocytes (A) LR73 cells were transfected with the indicated plasmids. After 1 day, cells were incubated with apoptotic thymocytes or live thymocytes for 10 min and lysed. FLAG-tagged Orai1 was immunoprecipitated with anti-FLAG antibody-conjugated agarose beads. Bound proteins were detected with the indicated antibodies (left) and co-immunoprecipitated STIM1 with Orai1 was quantified (right). The images are representative of three independent experiments. Mean ± SEM (two-tailed unpaired Student’s t test). (B) Lysates of BMDMs stimulated with apoptotic cells for 10 min were incubated with an anti-Orai1 antibody and protein A/G agarose beads. Bound proteins were detected by immunoblotting (left) and co-immunoprecipitated STIM1 with Orai1 was quantified (right). The arrow head indicates Orai1. The images are representative of three independent experiments. Mean ± SEM (two-tailed unpaired Student’s t test). (C,D) LR73 cells transfected with Orai1-CFP and STIM1-YFP were incubated with TAMRA-stained apoptotic cells for the indicated duration. FRET signals were observed by time-lapse confocal microscopy (C) and quantified (D). Images were obtained every 10 sec for 30 min. Scale bar, 10 µm. n = 12 (-Apop. thy) and n = 13 (+Apop. thy.) cells, mean ± SEM. (E) LR73 cells transfected with Orai1-CFP and STIM1-YFP were stimulated with apoptotic or living thymocytes. FRET signals were detected using a microplate reader. n = 3 experiments, mean ± SEM (two-way ANOVA).
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