Supplementary MaterialsFigure S1: Ganglioside structures. ganglioside-containing LUVs and VLPs in mDCs.
June 5, 2019
Supplementary MaterialsFigure S1: Ganglioside structures. ganglioside-containing LUVs and VLPs in mDCs. Confocal microscopy analysis of mDCs previously pulsed with 100 M of GM1a, GM2, and GM3 containing LUVHIV-tRed and then exposed to 75 ng of VLPHIV-Gag-eGFP Gag as in Figure 2B. 3-D reconstructions of the sections collected throughout the whole mDC volume every 0.1 m. Isosurface representation of DAPI stained nucleus is shown, computing the maximum intensity fluorescence within a 3-D volumetric data field, where VLPHIV-Gag-eGFP and ganglioside-containing LUVHIV-tRed polarized towards the same area of mDCs.(PDF) pbio.1001315.s004.pdf (776K) GUID:?DAF0D028-5A4D-4388-A2B7-A8CB94F221EF Figure S5: Binding of distinct LUVs to mDCs. (A) Comparative mDC binding of LUVHIV-tRed containing GM3, Cer, or PS treated or not with neuraminidase for 12 h prior to addition to cells. A total of 2105 DCs were pulsed for 20 min at 37C with 100 M of LUV, washed with PBS, and assessed by FACS to obtain the percentage of tRed-positive cells. Data show mean values and SEM of cells from three donors. mDCs bound significantly higher amounts of untreated GM3 containing LUVHIV-tRed than neuraminidase treated liposomes (test). (B) Binding pattern analysis of mDCs pulsed with LUVHIV-tRed containing GM3, Cer, or PS treated or not with neuraminidase for 12 h prior addition to cells. Cells were incubated for 20 min purchase Paclitaxel at 37C (top images) or 2 h at 16C (bottom images) with100 M of LUV, washed with PBS, and assessed by confocal microscopy. After 20 min at 37C, liposomes remained randomly bound and no evident polarization or internalization was detected, as seen in mDCs incubated at 16C to arrest endocytosis. Images show 3-D reconstruction of the sections collected throughout the whole mDC volume every 0.1 m, computing the maximum purchase Paclitaxel intensity fluorescence of the liposome red signal and DAPI-stained nucleus.(PDF) pbio.1001315.s005.pdf (1.1M) GUID:?DCC8D8E6-1F9A-4B2D-AD7A-291C3DCE68E1 Figure S6: Minimal energy structures of the different gangliosides tested. Blue shadow indicates the proposed sialyllactose viral attachment moiety recognized by mDCs. For comparative purposes, GM4 and Asialo GM1 lacking sialyllactose domains are also depicted.(PDF) pbio.1001315.s006.pdf (1.2M) GUID:?7F55C54A-7217-48F0-A1CC-82A92DE222DE Video S1: 3-D reconstruction of an mDC pulsed with GM3 containing LUVHIVtRed and then exposed to VLPHIV-Gag-eGFP. Confocal microscopy analysis of the mDC pulsed with GM3 including LUVHIVtRed and subjected to VLPHIV-Gag-eGFP as with Shape 2b. The video displays a 3-D reconstruction from the areas collected through the entire whole mDC quantity every 0.1 m. Isosurface representation of DAPI stained nucleus can LKB1 be depicted, computing the utmost intensity fluorescence from the sac-like area surface inside a 3-D volumetric data field, where VLPHIV-Gag-eGFP and GM3-including LUVHIV-tRed are gathered inside the same area.(MOV) pbio.1001315.s007.mov (1.4M) GUID:?E8712B03-D4DF-45E0-AE51-ABF9F7655EA6 Abstract HIV-1 is internalized into mature dendritic cells (mDCs) via an up to now undefined system with subsequent transfer of stored, infectious pathogen to CD4+ T lymphocytes. Therefore, HIV-1 subverts a DC antigen catch mechanism to market viral spread. Right here, we display that gangliosides within the HIV-1 membrane will be the crucial substances for mDC uptake. purchase Paclitaxel HIV-1 virus-like contaminants and liposomes mimicking the HIV-1 lipid structure were proven to work with a common internalization pathway as well as the same trafficking path within mDCs. Therefore, these total outcomes demonstrate that gangliosides can become viral connection elements, in addition with their popular function as mobile receptors for several infections. Furthermore, the sialyllactose molecule within particular gangliosides was defined as the determinant moiety for mDC HIV-1 uptake. Therefore, sialyllactose represents a book molecular recognition design for mDC catch, and may become important both for antigen demonstration resulting in immunity against pathogens as well as for succumbing to subversion by HIV-1. Writer Overview Antigen-presenting cells such as for example dendritic cells (DCs) must combat attacks, but infections including HIV possess evolved ways of evade their anti-viral activity. HIV can enter DCs with a noninfectious endocytic system and technique them into moving infectious virus on to bystander CD4+ T cells. Immature DC (iDCs) are characterized by high endocytic activity and low T-cell activation potential. Interestingly, several groups have shown that DCs that have undergone maturation (mDCs), a process that occurs on contact with a presentable antigen, capture higher numbers of HIV-1 particles than iDCs when they are matured in the presence of lipopolysaccharide. mDCs move to the lymph nodes where they have more opportunity to interact with T cells than.