Fig 2: Exosomes isolated from HIV-1 infected hPBMCs reduce the expression of p-DRP1, and p-eNOS in primary HBMVECs. a The schematic was indicated the culture, activation, infection, and isolation of exosomes from either HIV-1 infected (HIV+hPBMC Exo) or control hPBMCs. b Extracellular vesicles (EVs) were isolated from HIV-1 infected and uninfected hPBMCs, and the distribution of concentrations (particles/mL) with the particle diameter (nm) was measured by qNano-IZON system. c The concentration of exosomes isolated from HIV-1 infected and uninfected hPBMCs was compared. d The presence of HIV-1 p24 protein in control Exo (C) and HIV+hPBMC Exo was measured by ELISA. Representative western blot of e p-DRP1 and total DRP1, and f p-eNOS and total eNOS in cells exposed to control Exo and HIV+hPBMC Exo. g Quantitative analysis of p-DRP1, total DRP1, p-eNOS, and total eNOS. Error bars show mean ± SD, n = 3 independent experiments, and significant changes are presented as p values (*p < 0.05, **p < 0.003)

Fig 3: HIV-exosomes induce mitochondrial hyperfusion due to loss of p-Drp-1. a The effect of HIV-exosome on the mitochondrial fission and fusion events was studied by a co-localization experiment. After 24 h treatment with DiD-tagged (red) exosomes (25 µg/mL), cells were stained for 1 h with Hoechst 33342 nuclear dye (blue) and 45 min with MitoTracker Green (100 nM). After removal of Hoechst and MitoTracker Green, cells were washed with PBS, and the pictures were taken under fluorescence microscopy with ×60 magnification. For better image quality, the brightness and contrast were adjusted uniformly within experiments using the ImageJ software (version 1.50). b The mitochondrial size was measured by the ImageJ/Fiji software. More than 200 mitochondria were counted and compared from each of the given treatment conditions. The mitochondrial count was from at least three cells (n = 3) for each treatment condition. Error bars show mean ± SD, and significant changes are presented as p values (*p < 0.05, **p < 0.005). Cells were treated in a time-dependent manner as indicated, with representative western blot (c), and quantitative analysis (d) of p-DRP1 and total DRP1. In the second experiment, cells were treated for 24 h with increasing concentrations of U1 Exo. e Representative western blot and f quantitative analysis of p-DRP1 and total DRP1. Representative western blot (g), and quantitative analysis (h) of p-DRP1 and total DRP1 in cells exposed to HIV(-) Jurkat Exo. FCCP was used as positive control. Error bars show mean ± SD, n = 3 independent experiments, and significant changes are presented as p values

Fig 4: Shigella fragment mitochondria to counteract septin cage assembly HeLa cells stably expressing SEPT6-GFP were treated with Drp1 siRNA for 72 h, transfected with mito-BFP 24 h and then infected with Shigella flexneri-mCherry for 2 h for live confocal microscopy. Representative image shows interplay between two Shigella–septin cages and fused mitochondria. The scale bar represents 1 µm. See also Movie EV4.HeLa cells were treated with Drp1 siRNA for 72 h and then infected with x-light Shigella-mCherry for 4 h 40 min for quantitative confocal microscopy. IPTG was added 30 min prior to fixation, and then samples were labelled with antibody for SEPT7. Graph represents mean % ± SEM of Shigella responding to IPTG outside (-) or inside (+) SEPT7 cages from three independent experiments. Student's t-test, **P < 0.01.HeLa cells were infected with S. flexneri M90T for 4 h 40 min, labelled with MitoTracker Red CMXRos and fixed for quantitative confocal microscopy. Boxplots show the length of mitochondria (µm; whiskers from min to max) surrounding Shigella outside (-) or inside (+) SEPT7 cages from three independent experiments (analysis of at least 130 measurements per biological replicate). Student's t-test, ***P < 0.001.HeLa cells were either kept uninfected as a control or infected with S. flexneri M90T or S. flexneri ?IcsA for 4 h 40 min. Samples were labelled with MitoTracker Red CMXRos and fixed for quantitative confocal microscopy. Boxplots show length of mitochondria (µm; whiskers from min to max) in uninfected cells (CTRL), surrounding S. flexneri M90T (+M90T) or S. flexneri ?IcsA (+?IcsA) from three independent experiments (analysis of at least 250 measurements per biological replicate). Student's t-test, ***P < 0.001.A potential model for the Shigella–septin cage and actin tail assembly pathways. Based on our findings, we propose that (i) mitochondria promote septin cage assembly for antibacterial autophagy or (ii) Shigella fragment mitochondria to counteract septin cage entrapment. Depending on the fragmentation of mitochondria by IcsA, Shigella will be compartmentalised in septin cages or spread cell-to-cell via actin-based motility.

Fig 5: Drp1 and Parkin affects mitochondrial copy and PRRSV replicationA. Marc145 cells were transfected with negative control (NC) or specific siRNA targeting Drp1 or Parkin. At 24h post-transfection, the silencing effect was determined. B. and C. The DNA were extracted from Mock-, NC-, Drp1- and Parkin-transfected cells and subjected to mitochondrial copy determination by qPCR. D. and E. The effect of Drp1 and Parkin silencing on PRRSV replication were determined by Western Blot. Nsp2 protein gray was quantified by Image J. Data are expressed as means with SEM of three independent experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001.