Fig. 5: The role of F-ApoEVs in cell clearance and intercellular communication.

a Representative maximum intensity projection (MIP) images from time-lapse lattice light sheet microscopy (LLSM) of cell trace violet-stained BMDM interacting with and engulfing MEF-derived FOOD/F-ApoEVs stained with A5-PE (magenta). b Quantification of re-feeding engulfment assay measured by %Cypher5E⁺ BMDMs following initial incubation with the FOotprint Of Death (FOOD)/FOOD-derived ApoEVs (F-ApoEVs) derived from 1 ×10⁴ or 2 ×10⁴ MEFs for ~24 h, followed by incubation of CypHer5E-labelled apoptotic Jurkat T cells. Data is representative of (n = 3) independent experiments. c Representative time-lapse MIP confocal laser scanning microscopy (CLSM) images of FOOD formation in A549 cells 19- to 24 h post-infection (p.i.) with influenza A virus (IAV) (MOI = 10) (right). Quantification of the frequency of FOOD formation in IAV-infected apoptotic cells (left). Data is representative of (n = 3) independent experiments. d Representative scanning electron microscopy (SEM) images of FOOD formation in IAV-infected apoptotic cells (24 h p.i.). Data is representative of (n = 2) independent experiments. Localisation of viral nucleoprotein (NP) (e) or hemagglutinin (HA) (f) (green) in IAV-infected A549 cell-derived FOOD/ F-ApoEVs (24 h p.i.). Quantification of %FOOD stained positive for NP or HA protein shown on the right. g Representative transmission electron microscopy (TEM) images of isolated F-ApoEVs from A549 cells induced to undergo apoptosis with either BH3-mimetic cocktail treatment, or IAV infection. White arrows indicate virions. Quantification of the absolute number of NP⁺ A549 cells (h) or NP⁺A5⁺ A549 cells (i) following co-incubation of A549 cells with the indicated amount of F-ApoEVs isolated from BH3-mimetic cocktail treated or IAV infected A549 cells. Data is representative of (n = 3) independent experiments. Error bars represent s.e.m. Unpaired student’s two tailed t-test was performed to determine the indicated p-values.