Figure 3. FMRP deficiency increases efferocytosis in vivo .

• A–E
  In vitro and in vivo efferocytosis experiments, where percentage of macrophages F4/80⁺ (red) that ingested apoptotic cells (AC) labeled with carboxyfluorescein succinimidyl ester (CFSE)⁺ (green) were reported as % efferocytosis. (A) BMDMs were transfected with Fmr1‐ or control‐siRNA and incubated CFSE‐labeled AC for the indicated hours (n = 4 biological replicates). (B) Fmr1^+/+ and Fmr1^−/− BMDMs were treated with PA (500 µM) for 6 h and then incubated with CFSE‐labeled ACs for 4 h (n = 4 biological replicates). (C) Fmr1^+/+ and Fmr1^−/− mice were fed WD (16 weeks) and injected intraperitoneally with CFSE‐labeled AC (1.5 h), followed by PM elicitation (n = 4–5 mice per group). (D) BMDM were pre‐treated either with vehicle (DMSO) or AMG‐18 (5 µM) for 1 h then incubated with CFSE‐labeled Acs for 4 h (n = 3 biological replicates). (E) C57BL/6 mice were injected with AMG‐18 (30 mg/kg) or vehicle (DMSO) for 8 h, followed by intraperitoneal injection with CFSE‐labeled ACs for 1.5 h and PM elicitation (n = 4 mice per group).
• F, G
  In vitro continuous efferocytosis experiments, where macrophages were stained for F4/80⁺ (red), AC were labeled with CFSE (AC‐1; green) or Violet (AC2; violet). % continuous efferocytosis was determined by the ratio of F4/80⁺, CFSE⁺, and Violet⁺ (triple positive) cells to total F4/80⁺ and CFSE⁺ (double positive) cells. (F) Fmr1^+/+ and Fmr1^−/− BMDM were incubated with AC‐1 for 2 h, and after 2 h interval, incubated with AC‐2 for 2 more hours (n = 4–3 biological replicates). (G) BMDM were pre‐treated either with vehicle (DMSO) or AMG‐18 (5 µM) for 1 h, incubated with CFSE‐labeled AC‐1 for 2 h, followed by incubation with Violet‐labeled AC‐2 for 2 h and PM collection (n = 4 biological replicates).

Data information: For all images scale bar = 50 µm; Red: Macrophages, Green: AC/AC‐1, Violet: AC‐2. Data are mean ± SEM. Unpaired t‐test with Welch’s correction.

Source data are available online for this figure.