Fig 2: HIV-1 infection and/or modifications of the cav-1/ocln/Alix complex affect anti- and pro-inflammatory cytokine profile in pericytes. Pericytes were transfected with cav-1 siRNA (Cav-1-), ocln expression vector (Ocln+), or Alix siRNA (Alix-) as in Figure 5 and either mock-infected or HIV-1-infected for 48 or 72 h. Cytokine levels were analyzed in cell culture media by Bio-Plex Pro Human Cytokine assay kit. INF-? levels decreased to non-detectable (#) concentrations after HIV-1 infection in control, cav-1-, and Alix- pericyte cultures. The data are mean ± SEM from two independent experiments. ****P < .0001, ***P = .0002, **P = .003, *P < .0449, n = 4 per group

Fig 3: Modifications of the cav-1-ocln-Alix complex regulates HIV-1 infection. A, Pericytes were transfected with cav-1 siRNA (1 µg), ocln expression vector (2 µg), or/and Alix siRNA (1 µg) per 106 cells. When treatment with all three agents was combined, they were used at half the concentrations. Transfected cultures were then either mock-infected or HIV-1-infected as in Figure 1 and p24 was analyzed in cell culture media by ELISA. B, p24 levels in the media from pericyte cultures exposed to conditioned media from (A) for 48 h. C, Confocal microscopy images of HIV-1 pNL4-3-GFP-infected pericytes after ocln overexpression, cav-1 silencing, and Alix silencing. Nuclei were stained with DAPI (blue). GFP fluorescence (green) reflects HIV-1 transcription. D, Quantification of the mean fluorescence intensity (MFI) of GFP from (C). Data represents mean ± SEM from two independent experiments. ****P < .0001, ***P = .0002, **P = .003, *P < .0449, n = 4 per group. Scale bars, 20 µm

Fig 4: Cav-1, ocln, and Alix form a stable complex in mock-infected and HIV-1-infected pericytes. A, Diagram illustrating Alix and its binding partners identified in this study in a structural ribbon representation. The ESCRT-associated protein Alix binds tyrosine motifs via its C-terminal Proline Rich Domain (PRD). Caveolae are made up of oligomers of cav-1 and cavin proteins, and the figure shows a trimeric coiled coil for Cavin4a HR1 domain. For ocln, the region shown is the cytoplasmic C-terminal region that is known to bind scaffolding proteins. Figure was prepared using PyMol with the listed PDB accession numbers. B-E, Pericytes were either mock-infected (B) or infected with HIV-1 (C) for 48 h as in Figure 1. A total of 600 µg of cell lysate protein was incubated with cav-1 antibody for 24 h, followed by incubation with protein A/G PLUS-Agarose beads. Both immunoprecipitates and supernatants were analyzed by immunoblotting for the presence of cav-1, ocln, and Alix. D, Immunostaining of cav-1 (green), ocln (red), and Alix (purple) in mock-infected or HIV-1-infected for 48 h. E, Cellular localization of the cav-1, ocln, and Alix complex in mock-infected or HIV-1-infected for 48 h. Cell membranes are visualized by blue fluorescent dye DiB (left panel). Middle panel, the cav-1, ocln, and Alix complexes merged with a bright field image of a single pericyte. The images were analyzed as in Figure 1; n = 4 per group; scale bar 5, 10, or 20 µm

Fig 5: Extracellular vesicles released by HSC-3 cells have characteristics of exosomes and carry pain and metastasis genes as cargo. (a) Representative transmission electron micrographs of EVs isolated from conditioned media of HSC-3. Images were taken at 45,000× (left) and 92,00× (right) magnification. Isolated vesicles display cup-shaped morphology and membrane layer characteristic of exosomes (arrows). (b) The diameters of vesicles from HSC-3 were measured on micrographs taken at 92,000× (4–6 fields). (c) Representative nanoparticle tracking analysis (NTA) of the distribution of measured EV hydrodynamic diameter (mode indicated above the trace). Extracellular vesicle sizes as measured from electron micrographs and by NTA are typical of exosomes. (d) Isolated EVs express exosome endocytic marker proteins TSG101 and ALIX, and tetraspanin, CD63. Calnexin (CANX, endoplasmic reticulum marker) and GM130 (cis-Golgi network marker) were not detected. Exosome samples were obtained from cells grown under normoxic conditions. Pain and metastasis gene proteins, MMP1 and THBS1 were detected in the HSC-3 EVs.