Figure EV3. Formation of Annexin V‐positive membrane domains requires physical contact between Mtb aggregates and live macrophages.

• A, B
  Representative fluorescence microscopy images of cytochalasin D‐treated BMDMs infected with aggregates of Mtb Erd‐tdTomato in the presence of Annexin V‐FITC and fixed at 8 h post‐infection. The plasma membrane of the cells was stained with an anti‐CD‐45 antibody and nuclei were stained with Hoechst (white). Yellow arrows point at Mtb aggregates (magenta) overlapping with areas that stain positive for Annexin V (blue) and macrophages plasma membrane (yellow). White arrows indicate an Mtb aggregate that do not colocalize neither with the macrophages plasma membrane now with an Annexin V area. Cyan arrows indicate the distal area of an Mtb aggregate that stains positive for Annexin V but does not colocalize with the macrophages plasma membrane. Scale bars, 20 μm.
• C, D
  BMDMs treated with cytochalasin D were infected with aggregates of Mtb, incubated with Annexin V, and imaged by time‐lapse microscopy at 1‐h intervals for 60 h. (C) Example of bacterial aggregates (magenta, top panels) that do not interact with macrophages and never become Annexin V‐positive (yellow, bottom panel) during the course of the experiment. (D) Example of bacterial aggregate (magenta, top panels) that induces the formation of a local Annexin V‐positive membrane domain in the interacting macrophage (yellow, bottom panel). After the death of the macrophage (at 18:00 h) the bacterial aggregates gradually lose fluorescence (40:00 h–60:00 h). Scale bars, 20 μm.