Fig. 4: Formation of FOOD and F-ApoEVs is dependent on caspase-cleaved ROCK1. | Nature Communications

Fig. 4: Formation of FOOD and F-ApoEVs is dependent on caspase-cleaved ROCK1.

From: The formation of the ‘footprint of death’ as a mechanism for generating large substrate-bound extracellular vesicles that mark the site of cell death

Fig. 4

a WT or ROCK1nc MEFs were treated with a BH3-mimetic cocktail (5 μM ABT-737, 10 μM S63845) and imaged by time-lapse confocal laser scanning microscopy (CLSM). Cell membrane and nucleus were visualised by PHK26 and Hoechst 33342 staining, respectively, and exposed phosphatidylserine (PtdSer) with A5-FITC. b Quantification of the number of FOOD-derived ApoEV (F-ApoEVs) generated per cell from CLSM imaging, by WT (grey) or ROCK1nc (blue) MEFs, 60-, 120-, and 180 min post initial PtdSer exposure. Data points represent individual cells (n = 12). c Quantitative analysis of The FOotprint Of Death (FOOD) generated by WT or ROCK1nc MEFs: number of branches per cell, area of branches per cell (μm2), and branch thickness (μm). Data is pooled from (n = 3) independent experiments. d Representative scanning electron microscopy (SEM) images of WT or ROCK1nc MEFs, treated with a BH3-mimetic cocktail. (n = 2). e Visualisation of actin in FOOD/F-ApoEV formation in WT or ROCK1nc MEFs following treatment with a BH3-mimetic cocktail. Representative maximum intensity projection (MIP) images from time-lapse CLSM shown. Cell actin was visualised by SiR-Actin and exposed PtdSer with A5-FITC. Error bars in (b, c) represent s.e.m. Unpaired student’s two tailed t-test was performed to determine the indicated p-values.

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