Journal: Nature communications

Article Title: Syntenin-ALIX exosome biogenesis and budding into multivesicular bodies are controlled by ARF6 and PLD2.

doi: 10.1038/ncomms4477

Figure Lengend Snippet: Figure 5 | ARF6 depletion does not affect viral budding. (a) Western blot illustrating sustained ARF6 depletion during the time course of the experiment, viral release being measured 6 days after RNAi treatment. (b,c) MCF-7 cells infected with VSV-G-pseudotyped HIV-1 were analysed by confocal fluorescence microscopy to test for their ability to allow viral budding. (b) Viral envelope proteins were stained with anti-Env SUgp120 antibodies (green) and viruses that completed their budding were detected with an antibody recognizing mature matrix protein MAp17 (red). Note the high level of colocalization illustrating that most virions completed their budding at the plasma membrane. Scale bar, 5 mm. Nuclei were stained with DAPI (blue). (c) The total MAp17 fluorescence was divided by the total number of nuclei in each field, calculated for 150 to 200 cells in three independent experiments. Histograms represent the mean fluorescence intensity per nucleus±s.d. as an estimation of the number of mature viruses released per cell. NS, non significant (Student’s t-test). (d) After RNAi treatment, MCF-7 cells were infected with VSV-G-pseudotyped HIV-1. Three days after infection, cells were analysed by confocal fluorescence microscopy using a p24 antibody to stain intracellular Gag and the mature virus. The total p24 fluorescence was divided by the total number of nuclei in each field, calculated for 150 to 200 cells in three independent experiments. Histograms represent the mean fluore- scence intensity per nucleus±s.d. NS, non significant (Student’s t-test). (e) Virus release was determined by CAp24 quantification in the supernatant, using a HIV-1 p24 ELISA kit. p24 release was divided by the total number of cell. Results were normalized to 100% in siCNT cells. NS, non significant (Student’s t-test).

Article Snippet: Virus concentration was determined by CAp24 quantification in the supernatant, using a HIV-1 p24 ELISA kit (PerkinElmer Life Science, Inc.).

Techniques: Western Blot, Infection, Microscopy, Staining, Clinical Proteomics, Membrane, Virus, Enzyme-linked Immunosorbent Assay

Journal: Nature cancer

Article Title: Targeting immunosuppressive macrophages overcomes PARP inhibitor resistance in BRCA1-associated triple-negative breast cancer

doi: 10.1038/s43018-020-00148-7

Figure Lengend Snippet: Antibodies used for CyCIF

Article Snippet: Error bars represent standard error of the mean (±SEM). table ft1 table-wrap mode="anchored" t5 Table 1. caption a7 Cycle Channel Antibody Fluorophore Manufacturer Clone Catalog Number Dilution Cycle1 FITC anti-Rabbit-488 Alexa Fluor 488 ThermoFisher Polyclonal A-21246 1:2000 Cy3 anti-Rat-555 Alexa Fluor 555 ThermoFisher Polyclonal A-21434 1:2000 Cy5 anti-Mouse-647 Alexa Fluor 647 ThermoFisher Polyclonal A-21237 1:2000 Cycle 2 FITC anti-GZMB anti-Rabbit-488 Abcam Polyclonal AB4059 1:500 Cy3 anti-GZMB anti-Rat-555 eBioscience 496B 14–8889-80 1:250 Cy5 anti-GZMB anti-Mouse-647 Dako GrB-7 M7235 1:200 Cycle 3 FITC CD8a-488 Alexa Fluor 488 eBioscience AMC908 53–0008-80 1:400 Cy3 CD3D-555 Alexa Fluor 555 Abcam EP4426 {"type":"entrez-nucleotide","attrs":{"text":"AB208514","term_id":"84781018","term_text":"AB208514"}} AB208514 1:200 Cy5 CD45–647 Alexa Fluor 647 BioLegend HI30 304020 1:200 Cycle 4 FITC CD68–488 Alexa Fluor 488 CST D4B9C 24850S 1:200 Cy3 Foxp3–570 eFluor 570 eBioscience 236A/E7 41–4777-82 1:150 Cy5 CD14–647 Alexa Fluor 647 Abcam Polyclonal ab196169 1:400 Cycle 5 FITC CD163–488 Alexa Fluor 488 Abcam {"type":"entrez-protein","attrs":{"text":"EPR14643","term_id":"523380394","term_text":"EPR14643"}} EPR14643 –36 ab218293 1:200 Cy3 Keratin-570 eFluor 570 eBioscience AE1/AE3 41–9003-82 1:400 Cy5 PD1–647 Alexa Fluor 647 Abcam EPR4877(2) ab201825 1:200 Cycle 6 FITC CD4–488 Alexa Fluor 488 R&D Systems Polyclonal FAB8165G 1:200 Cy3 Ki67–570 eFluor 570 eBioscience 20Raj1 41–5699-80 1:200 Cy5 PDL1–647 Alexa Fluor 647 CST E1L3N 15005 1:200 Open in a separate window Antibodies used for CyCIF In BRCA1 -associated TNBC, macrophages were strikingly the most abundant immune cell population in the tumor, as demonstrated by a significant enrichment of CD68 + and CD163 + cells.

Techniques:

Journal: Nature cancer

Article Title: Targeting immunosuppressive macrophages overcomes PARP inhibitor resistance in BRCA1-associated triple-negative breast cancer

doi: 10.1038/s43018-020-00148-7

Figure Lengend Snippet: Antibodies used for murine flow cytometry

Article Snippet: Error bars represent standard error of the mean (±SEM). table ft1 table-wrap mode="anchored" t5 Table 1. caption a7 Cycle Channel Antibody Fluorophore Manufacturer Clone Catalog Number Dilution Cycle1 FITC anti-Rabbit-488 Alexa Fluor 488 ThermoFisher Polyclonal A-21246 1:2000 Cy3 anti-Rat-555 Alexa Fluor 555 ThermoFisher Polyclonal A-21434 1:2000 Cy5 anti-Mouse-647 Alexa Fluor 647 ThermoFisher Polyclonal A-21237 1:2000 Cycle 2 FITC anti-GZMB anti-Rabbit-488 Abcam Polyclonal AB4059 1:500 Cy3 anti-GZMB anti-Rat-555 eBioscience 496B 14–8889-80 1:250 Cy5 anti-GZMB anti-Mouse-647 Dako GrB-7 M7235 1:200 Cycle 3 FITC CD8a-488 Alexa Fluor 488 eBioscience AMC908 53–0008-80 1:400 Cy3 CD3D-555 Alexa Fluor 555 Abcam EP4426 {"type":"entrez-nucleotide","attrs":{"text":"AB208514","term_id":"84781018","term_text":"AB208514"}} AB208514 1:200 Cy5 CD45–647 Alexa Fluor 647 BioLegend HI30 304020 1:200 Cycle 4 FITC CD68–488 Alexa Fluor 488 CST D4B9C 24850S 1:200 Cy3 Foxp3–570 eFluor 570 eBioscience 236A/E7 41–4777-82 1:150 Cy5 CD14–647 Alexa Fluor 647 Abcam Polyclonal ab196169 1:400 Cycle 5 FITC CD163–488 Alexa Fluor 488 Abcam {"type":"entrez-protein","attrs":{"text":"EPR14643","term_id":"523380394","term_text":"EPR14643"}} EPR14643 –36 ab218293 1:200 Cy3 Keratin-570 eFluor 570 eBioscience AE1/AE3 41–9003-82 1:400 Cy5 PD1–647 Alexa Fluor 647 Abcam EPR4877(2) ab201825 1:200 Cycle 6 FITC CD4–488 Alexa Fluor 488 R&D Systems Polyclonal FAB8165G 1:200 Cy3 Ki67–570 eFluor 570 eBioscience 20Raj1 41–5699-80 1:200 Cy5 PDL1–647 Alexa Fluor 647 CST E1L3N 15005 1:200 Open in a separate window Antibodies used for CyCIF In BRCA1 -associated TNBC, macrophages were strikingly the most abundant immune cell population in the tumor, as demonstrated by a significant enrichment of CD68 + and CD163 + cells.

Techniques:

Journal: Nature cancer

Article Title: Targeting immunosuppressive macrophages overcomes PARP inhibitor resistance in BRCA1-associated triple-negative breast cancer

doi: 10.1038/s43018-020-00148-7

Figure Lengend Snippet: Antibodies used for human flow cytometry

Article Snippet: Error bars represent standard error of the mean (±SEM). table ft1 table-wrap mode="anchored" t5 Table 1. caption a7 Cycle Channel Antibody Fluorophore Manufacturer Clone Catalog Number Dilution Cycle1 FITC anti-Rabbit-488 Alexa Fluor 488 ThermoFisher Polyclonal A-21246 1:2000 Cy3 anti-Rat-555 Alexa Fluor 555 ThermoFisher Polyclonal A-21434 1:2000 Cy5 anti-Mouse-647 Alexa Fluor 647 ThermoFisher Polyclonal A-21237 1:2000 Cycle 2 FITC anti-GZMB anti-Rabbit-488 Abcam Polyclonal AB4059 1:500 Cy3 anti-GZMB anti-Rat-555 eBioscience 496B 14–8889-80 1:250 Cy5 anti-GZMB anti-Mouse-647 Dako GrB-7 M7235 1:200 Cycle 3 FITC CD8a-488 Alexa Fluor 488 eBioscience AMC908 53–0008-80 1:400 Cy3 CD3D-555 Alexa Fluor 555 Abcam EP4426 {"type":"entrez-nucleotide","attrs":{"text":"AB208514","term_id":"84781018","term_text":"AB208514"}} AB208514 1:200 Cy5 CD45–647 Alexa Fluor 647 BioLegend HI30 304020 1:200 Cycle 4 FITC CD68–488 Alexa Fluor 488 CST D4B9C 24850S 1:200 Cy3 Foxp3–570 eFluor 570 eBioscience 236A/E7 41–4777-82 1:150 Cy5 CD14–647 Alexa Fluor 647 Abcam Polyclonal ab196169 1:400 Cycle 5 FITC CD163–488 Alexa Fluor 488 Abcam {"type":"entrez-protein","attrs":{"text":"EPR14643","term_id":"523380394","term_text":"EPR14643"}} EPR14643 –36 ab218293 1:200 Cy3 Keratin-570 eFluor 570 eBioscience AE1/AE3 41–9003-82 1:400 Cy5 PD1–647 Alexa Fluor 647 Abcam EPR4877(2) ab201825 1:200 Cycle 6 FITC CD4–488 Alexa Fluor 488 R&D Systems Polyclonal FAB8165G 1:200 Cy3 Ki67–570 eFluor 570 eBioscience 20Raj1 41–5699-80 1:200 Cy5 PDL1–647 Alexa Fluor 647 CST E1L3N 15005 1:200 Open in a separate window Antibodies used for CyCIF In BRCA1 -associated TNBC, macrophages were strikingly the most abundant immune cell population in the tumor, as demonstrated by a significant enrichment of CD68 + and CD163 + cells.

Techniques:

Journal: EMBO molecular medicine

Article Title: R5 HIV-1 envelope attracts dendritic cells to cross the human intestinal epithelium and sample luminal virions via engagement of the CCR5.

doi: 10.1002/emmm.201202232

Figure Lengend Snippet: Figure 1. R5 but not X4 HIV-1 induces DCs to migrate through a monolayer of epithelial cells. Caco-2 cells were grown on transwell filter to form a confluent monolayer then DCs were let to adhere to the bottom of the filter. Cell-free HIV-1, LPS (1 mg/ml) or medium (DMEM 10% FCS) were incubated on the apical side of the Caco-2 monolayer for 1.5 h. Filters were processed for CM. A–F. Three-dimensional rendering of representative fields obtained with Volocity 5.0 software. Mouse anti-human E-Cadherin þ Alexafluor488 goat anti-mouse IgG2a (green) depicts interepithelial adherent junctions. Mouse anti-human DC-SIGN-PE (red) labels the DCs, which creep through epithelial cells in response to incubation with R5 HIV-1J6363 (A, at 1 ng of p24 Ag) and HIV-1AD8 (B, at 24 ng of p24 Ag) but do not in response to X4 HIV-1IIIB (D, at 10 ng of p24 Ag) and HIV-1pNL4.3 (E, at 40 ng of p24 Ag). LPS (C) and medium (F) were positive and negative controls. The experiment was repeated three times. G,H. Quantitative analysis of DCs migration across the Caco-2 cell monolayer at the apical (G) and medial (H) level of the cell layer is shown. Results are expressed as percentage of area occupied by DCs compared to that of the whole field. Bars represent mean SD of three or four fields of three different experiments. Statistic analysis was performed as described in Materials and Methods Section. p < 0.05.

Article Snippet: Culture supernatants were collected at consecutive time points up to 21 days and viral growth determined by an HIV-1 p24 Ag ELISA (lower detection limit 4 pg/ml, Aalto Bioreagents, www.aaltobioreagents.ie).

Techniques: Incubation, Software, Migration

Journal: EMBO molecular medicine

Article Title: R5 HIV-1 envelope attracts dendritic cells to cross the human intestinal epithelium and sample luminal virions via engagement of the CCR5.

doi: 10.1002/emmm.201202232

Figure Lengend Snippet: Figure 2. DCs migration does not alter junctional protein expression by Caco-2 cells and preserves the integrity of the HIV-1 treated monolayer. The Caco-2/DCs co-culture system was incubated with medium or R5 HIV-1AD8 at 24 ng of p24 Ag for 1.5 h. A–D. CM cross-sectional images of specimens stained for DCSIGN-PE (red) and for the epithelial junctions (green) JAM (A), E-Cadherin (B) Occludin (C) ZO-1 (D) showed that HIV-1 as well as migrated DCs did not affect the intraepithelial junctions expression in the Caco-2/DC system. DAPI stained the nuclei. Results are from one representative experiment out of three. E. Ultrastructure of Caco-2/DCs culture treated with R5 HIV-1J6363 (at 1 ng of p24 Ag) for 1.5 h. TEM images show DCs adhering to the filter (), DCs protrusions inside the filter membrane pore () and a DC interposed between adjacent Caco-2 cells () Scale bar: 2 mm. Numbers identify the corresponding magnified images displayed in panel (E), and arrows point to interepithelial TJs (1, 2), TJs-like structures between DCs and Caco-2 cells (3–5), contiguity among cells inside the pore (6), and junction like-structures between DCs (7) Scale bar: 500 nm. Results from a representative experiment out of three are shown. F. Semi-thin sections for TEM labelled with Toluidine blue and the corresponding explicative colour mask below (Caco-2 cells red, DCs blue, filter grey) show the morphology and the spatial organization of cells. Caco-2 cells are columnar and polarized, displaying microvilli, dense cytoplasmic granules and vacuoles characteristic of epithelial cells. DCs are disposed along the lower face of the filter, inside the membrane pores and, in HIV-1 treated sample, intercalated in between Caco-2 cells, without destroying epithelial monolayer continuity. G. The permeability of the Caco-2/DCs culture to FD4 (4 kDa, 250 mg/ml) in the presence of medium, R5 HIV-1AD8 (at 24 ng of p24 Ag) and CN54 gp140 protein (100 ng/ml) is shown as percentage of the positive control (i.e. FD4 added in the upper chamber of the transwell without Caco-2 cells). Triton X100 was included as control of barrier disruption. Results are mean values SD of triplicates from a representative experiment out of three.

Article Snippet: Culture supernatants were collected at consecutive time points up to 21 days and viral growth determined by an HIV-1 p24 Ag ELISA (lower detection limit 4 pg/ml, Aalto Bioreagents, www.aaltobioreagents.ie).

Techniques: Migration, Expressing, Co-Culture Assay, Incubation, Staining, Membrane, Permeability, Positive Control, Control, Disruption

Journal: EMBO molecular medicine

Article Title: R5 HIV-1 envelope attracts dendritic cells to cross the human intestinal epithelium and sample luminal virions via engagement of the CCR5.

doi: 10.1002/emmm.201202232

Figure Lengend Snippet: Figure 4. R5 HIV-1 induces migration of DCs through the colonic epithelium. Colonic tissue was either left untreated (A and B) or incubated with X4 HIV-1pNL4.3 (C), R5 HIV-1AD8 (D) or R5 HIV-1J6363 (E–J) (at 50 ng of p24 Ag) for 30 min. CD11cþ cells were detected in the colonic lamina propria of untreated (B) and HIV-1pNL4.3 treated (C) tissues but not in between epithelial cells. Following R5 HIV-1 incubation protruded dendrites (D) or whole DCs (E–H) were observed inside the epithelium. Moreover DCs (DCSIGNþ/CD68

Article Snippet: Culture supernatants were collected at consecutive time points up to 21 days and viral growth determined by an HIV-1 p24 Ag ELISA (lower detection limit 4 pg/ml, Aalto Bioreagents, www.aaltobioreagents.ie).

Techniques: Migration, Incubation

Journal: EMBO molecular medicine

Article Title: R5 HIV-1 envelope attracts dendritic cells to cross the human intestinal epithelium and sample luminal virions via engagement of the CCR5.

doi: 10.1002/emmm.201202232

Figure Lengend Snippet: Figure 5. DCs capture HIV-1 in colonic explant and in vitro Caco-2/DC model. Human colonic tissue was either left untreated (A) or incubated with R5 HIV-1J6363 (B) or R5 HIV-1AD8 (C and D) (at 50 ng of p24 Ag) for 30 min. HIV-1 virions co-localized with CD11cþ DCs that migrate inside the epithelium as well as with DCs closely underlying the epithelium (see white arrows in B and C). Moreover virions were detected at both the apical and basal side as well as penetrating the epithelium. Yellow arrows point to virions entrapped in the mucus. Absence of p24 Ag in the basal medium, determined with ELISA, confirmed the seal integrity of the tissue culture system without any viral leakage (as described in Materials and Methods Section). Panel (D) is a magnification (zoom 3) of the boxed area in C. Cryosections were fixed with 4% PFA and stained with mouse anti-human CD11c-PE (DCs; red), mouse anti-p24 þ Alexafluor488 goat anti-mouse IgG (HIV-1; green), and DAPI (nuclei; blue). Scale bar indicate the magnifications. Each Figure is representative of results from three donor tissues. Caco-2/DCs co-culture was incubated with R5 HIV-1AD8-GFP (at 20 ng of p24 Ag) for 30 min (E), 1.5 h (F) or 4.5 h (G). (A and B) Three-dimensional renderings from CM z-series stacks of representative images (acquired with objective 40, zoom 6.5) showed GFP-expressing virions (green, indicated by arrows) either on migrated (E) as well as non- migrated DCs at the basal side (F, the bottom side of the filter is shown). Cells were stained for DAPI (all cells; blue) and mouse anti-human CD11c þ AlexaFluor594 goat anti-mouse IgG (DCs; red). (G) Three-dimensional rendering from a CM z-series stack of images showed a cluster of DCs (visualized with mouse anti-human DC-SIGN-PE; red) migrated to the Caco-2 side of the culture (Caco-2 cells not shown in the Figure) after incubation with HIV-1 AD8–GFP. GFP-expressing virions (yellow) were associated to migrated DCs.

Article Snippet: Culture supernatants were collected at consecutive time points up to 21 days and viral growth determined by an HIV-1 p24 Ag ELISA (lower detection limit 4 pg/ml, Aalto Bioreagents, www.aaltobioreagents.ie).

Techniques: In Vitro, Incubation, Enzyme-linked Immunosorbent Assay, Staining, Co-Culture Assay, Expressing

Journal: EMBO molecular medicine

Article Title: R5 HIV-1 envelope attracts dendritic cells to cross the human intestinal epithelium and sample luminal virions via engagement of the CCR5.

doi: 10.1002/emmm.201202232

Figure Lengend Snippet: Figure 7. DCs migration is dependent from the viral envelope. In the absence of the HIV-1 env as well as of the V1V3 env region, DCs did not migrate across the Caco-2 monolayer (A and B). The V1V3 env region of an R5 virus completely restored the migratory ability of DCs (C). A–C. The Caco-2/DCs co-culture was incubated apically for 1.5 h with HIV-1 deleted of the env (HIV-1Denv, 6 ng of p24 Ag) (A), the HIV-143DV deleted of the V1V3 region (40 ng of p24 Ag) as negative control (B) and a recombinant HIV-1 with the V1V3 env region of the R5 HIV-1J6363 primary virus recombined with the HIV-143DV backbone (HIV-1J6363-43DV, 20 ng of p24 Ag) (C). Three-dimensional reconstructions from CM z-series image stacks of the Caco-2/DCs culture stained for DAPI (epithelial cells and DCs; blue) and mouse anti-human DC-SIGN-PE (DCs; red) are shown. D,E. The monolayer of the Caco-2/DCs system was incubated for 1.5 h with medium (negative control), HIV-1YU2 (positive control) or YU2 gp120 protein (100 ng/ml). Bar charts represent quantitative analysis of DCs migration across the Caco-2 cell at the apical (D) and medial (E) level of the cell monolayer (as in Fig 1). Results are expressed as percentage of area occupied by DCs compared to that of the whole field. Bars represent mean SD of three different fields of three different experiments. Statistic analysis was performed as described in Materials and Methods Section. p < 0.05.

Article Snippet: Culture supernatants were collected at consecutive time points up to 21 days and viral growth determined by an HIV-1 p24 Ag ELISA (lower detection limit 4 pg/ml, Aalto Bioreagents, www.aaltobioreagents.ie).

Techniques: Migration, Virus, Co-Culture Assay, Incubation, Negative Control, Recombinant, Staining, Positive Control

Journal: EMBO molecular medicine

Article Title: R5 HIV-1 envelope attracts dendritic cells to cross the human intestinal epithelium and sample luminal virions via engagement of the CCR5.

doi: 10.1002/emmm.201202232

Figure Lengend Snippet: Figure 9. HIV-1 penetrates within and in between epithelial cells. A. Virions (stained with mouse anti-p24 þ Alexafluor488 goat anti-mouse IgG, green) were mainly localized at the apical surface of Caco-2 cells at 30 min but also inside the cytoplasm at 90 min of incubation with R5 HIV-1AD8 (20 ng of p24 Ag). Shown are CM single plane cross sectional images of a Caco-2 monolayer (rabbit anti-human Occludin þ Alexafluor594 goat anti-rabbit IgG; red) taken at apical and medial level of the cell layer. B. R5 HIV-1AD8 (red; visualized with human anti-gp120 monoclonal antibody 2G12 þ Alexafluor594 goat anti-human IgG) localized inside the cyto- plasm of epithelial cells and in intrajunctional spaces (indicated by arrows). Shown are four single plane cross sectional images, taken from the apical to the medial plane along the z-axis of the Caco-2 monolayer (mouse anti-human E-Cadherin þ Alexafluor488 rabbit anti-mouse IgG2a; green) incubated with R5 HIV-1J6363 (at 5 ng of p24 Ag) for 90 min. Results are of one representative experiment out of three.

Article Snippet: Culture supernatants were collected at consecutive time points up to 21 days and viral growth determined by an HIV-1 p24 Ag ELISA (lower detection limit 4 pg/ml, Aalto Bioreagents, www.aaltobioreagents.ie).

Techniques: Staining, Incubation

Journal: EMBO molecular medicine

Article Title: R5 HIV-1 envelope attracts dendritic cells to cross the human intestinal epithelium and sample luminal virions via engagement of the CCR5.

doi: 10.1002/emmm.201202232

Figure Lengend Snippet: Figure 10. Transcytosis of cell-free HIV-1 through a tight monolayer of Caco-2 cells. A–E. R5 HIV-1J6363 (A, at 1 ng of p24 Ag), X4 HIV-1IIIB (B, at 10 ng of p24 Ag), R5 93BR029 gp140 protein (C, at 100 ng/ml), R5 HIV-1SF162 (D, at 2 ng of p24 Ag), or medium (E), were added to the Caco-2/DCs cultures for 90 min and immunostained for CM analysis. HIV-1 (red; visualized with human anti-gp120 monoclonal antibody 2G12 þ Alexafluor594 goat-anti-human IgG) was dispersed throughout the cytoplasm but mainly concentrated in the upper part of the Caco-2 cells (visualized with mouse anti-human JAM þ Alexafluor 488 goat-anti-mouse IgG; green). DCs are not shown in the Figure. Transversal xy- and xz-plane visualization from representative fields of the monolayer were obtained with Volocity 5.0 software. 1 unit ¼ 10.3 mm. Results show one representative experiment out of three. F. Transcytosis of HIV-1 R5 and X4 is comparable. The amount of transcytosed virus was evaluated measuring with ELISA the p24 Ag released in the basal chamber after 2.5 h of incubation with cell-free HIV-1 (at 20 ng of p24 Ag), either R5 HIV-1AD8 or X4 HIV-1pNL4.3, on the apical side of the Caco-2 monolayer cultured with or without DCs adherent to the filter. Results are expressed as mean SD of triplicates of three different experiments.

Article Snippet: Culture supernatants were collected at consecutive time points up to 21 days and viral growth determined by an HIV-1 p24 Ag ELISA (lower detection limit 4 pg/ml, Aalto Bioreagents, www.aaltobioreagents.ie).

Techniques: Software, Virus, Enzyme-linked Immunosorbent Assay, Incubation, Cell Culture

Journal: EMBO molecular medicine

Article Title: R5 HIV-1 envelope attracts dendritic cells to cross the human intestinal epithelium and sample luminal virions via engagement of the CCR5.

doi: 10.1002/emmm.201202232

Figure Lengend Snippet: Figure 11. DCs capture virus and transfer infection to target cells. A,B. Caco-2/DCs system was apically incubated for 1.5 h with R5 HIV-1AD8 at 10 or 40 ng (A) or 20 ng of p24 Ag (B). PBMCs (5 105 cells) directly incubated with the same amount of input virus served as positive control (data not shown). (A) R5 virus does efficiently replicated when DCs were cultured with PBMCs (1 106). Low levels of p24 Ag were observed when DCs were cultured alone (inset). (B) DCs transmit infectious virus in the long-term. PBMCs were added to detached DCs immediately or after 3 or 4 days. Results from a representative experiment out of three are expressed as mean of p24 Ag values of triplicate cultures SD. C. DCs transferred both R5 and X4 viruses to PBMCs. Caco-2 cells were treated with R5 HIV-1AD8 (24 ng/ml) and X4 HIV-1pNL4.3 (40 ng/ml) as described above. DCs collected from transwell were co-cultured with PHA-activated PBMCs. Mean SD of three different experiments per- formed in triplicate is shown. p < 0.05.

Article Snippet: Culture supernatants were collected at consecutive time points up to 21 days and viral growth determined by an HIV-1 p24 Ag ELISA (lower detection limit 4 pg/ml, Aalto Bioreagents, www.aaltobioreagents.ie).

Techniques: Virus, Infection, Incubation, Positive Control, Cell Culture

Journal: iScience

Article Title: Sustained type I interferon signaling after human immunodeficiency virus type 1 infection of human iPSC derived microglia and cerebral organoids

doi: 10.1016/j.isci.2024.109628

Figure Lengend Snippet: HIV-1-infected microglia exhibit productive infection, activation, and dysregulated cytokine/chemokine production (A) RT-qPCR analysis of viral gag/pol mRNA after infection with HIV-1 JRFL in microglia monocultures; n = 3 independent infections of 03SF line iPSC-derived microglia; data are presented as the mean ± SEM. (B) ELISA analysis of secreted p24 in the culture supernatant of microglia infected with HIV-1 JR-FL reveals productive infection; n = 3 independent infections of 03SF iPSC-derived microglia; data are presented as the mean ± SEM. (C) RT-qPCR analysis of IL1 β , TNF α and IL-6 mRNA at days 16 and 23 post infection with HIV-1 JRFL; n = 3 independent infections of 03SF iPSC-derived microglia; two-way ANOVA; Šídák multiple comparisons test; ∗ p < 0.05; data are presented as the mean ± SEM. (D) RT-qPCR analysis of CCL2 , CCL3 , and CCL5 mRNA at days 16 and 23 post infection with HIV-1 JRFL; n = 3 independent infections of 03SF iPSC-derived microglia; two-way ANOVA; Šídák multiple comparisons test; ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001; data are presented as the mean ± SEM. (E) RT-qPCR analysis of IBA1 and P2RY12 mRNA at days 16 and 23 post infection with HIV-1 JRFL; n = 3 independent infections of 03SF iPSC-derived microglia; two-way ANOVA; Šídák multiple comparisons test; ∗ p < 0.05; data are presented as the mean ± SEM. (F) Luminex cytokine/chemokine analysis of secreted M-CSF and IP-10/CXCL10 culture supernatant from HIV-1 JRFL-infected microglia; n = 3 independent infections of 03SF microglia, two-way ANOVA or Mixed-effects model, respectively; Šídák or Holm-Šídák multiple comparisons test, respectively; ∗ p < 0.05; data are presented as the mean ± SEM. (G) Representative confocal images of nucleocapsid protein p24 staining in HIV-1 JRFL-infected 03SF-derived microglia (IBA1 + ). Scale bars, 10 μm.

Article Snippet: The amount of viral p24 was determined using the HIV-1 Gag p24 DuoSet ELISA (R &D System, DY7360-05).

Techniques: Infection, Activation Assay, Quantitative RT-PCR, Derivative Assay, Enzyme-linked Immunosorbent Assay, Luminex, Staining

Journal: iScience

Article Title: Sustained type I interferon signaling after human immunodeficiency virus type 1 infection of human iPSC derived microglia and cerebral organoids

doi: 10.1016/j.isci.2024.109628

Figure Lengend Snippet: HIV-1-infected microglia exhibit productive infection, activation, and dysregulated cytokine/chemokine production (A) RT-qPCR analysis of viral gag/pol mRNA at days 9, 16, 23, and 30 post infection with HIV-1 JRFL and HIV-1 YU2; two-way ANOVA; ∗ p < 0.05, ∗∗ p < 0.01; n = 3 independent infections of CD06 iPSC-derived microglia; data are presented as the mean ± SEM. (B) ELISA analysis of secreted p24 in the culture supernatant of microglia infected with HIV-1 JRFL and HIV-1 YU2 at days 9, 15, 21, and 27 reveals productive infection; n = 3 independent infections of CD06 iPSC-derived microglia; two-way ANOVA; ∗∗∗∗ p < 0.0001; data are presented as the mean ± SEM. (C) RT-qPCR analysis of IL1 β and TNFα mRNA at days 9, 16, 23, and 30 post infection with HIV-1 JRFL and HIV-1 YU2; n = 3 independent infections of CD06 iPSC-derived microglia; two-way ANOVA; Holm-Šídák multiple comparisons test; ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001; data are presented as the mean ± SEM. (D) RT-qPCR analysis of CCL2 and CCL3 mRNA at days 9, 16, 23, and 30 post infection with HIV-1 JRFL and HIV-1 YU2; n = 3 independent infections of CD06 iPSC-derived microglia; two-way ANOVA; Holm-Šídák multiple comparisons test; ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001; data are presented as the mean ± SEM. (E) RT-qPCR analysis of IBA1 and P2RY12 mRNA at days 9, 16, 23, and 30 post infection with HIV-1 JRFL and HIV-1 YU2; n = 3 independent infections of CD06 iPSC-derived microglia; two-way ANOVA; Holm-Šídák multiple comparisons test; ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001; data are presented as the mean ± SEM.

Article Snippet: The amount of viral p24 was determined using the HIV-1 Gag p24 DuoSet ELISA (R &D System, DY7360-05).

Techniques: Infection, Activation Assay, Quantitative RT-PCR, Derivative Assay, Enzyme-linked Immunosorbent Assay

Journal: iScience

Article Title: Sustained type I interferon signaling after human immunodeficiency virus type 1 infection of human iPSC derived microglia and cerebral organoids

doi: 10.1016/j.isci.2024.109628

Figure Lengend Snippet: HIV-1-infected microglia incorporate into sliced cerebral organoids (A) Representative confocal images of a day 144 organoid with neurons (MAP2 + ), astrocytes (GFAP + ), and microglia (IBA1 + ). Scale bars, 250 μm. (B) Representative confocal images of day 144 organoids 14 days after the addition of HIV-1 JRFL-infected or mock-infected microglia; (B1) shows a high-magnification image of HIV-1-infected microglia from (B). Scale bars, 250 μm or 20 μm as indicated. (C) High-magnification image of a single HIV-1-infected microglial cell within a sliced organoid. Scale bars, 20 μm. (D) RT-qPCR of gag/pol mRNA from HIV-1 JRFL-infected or mock-infected microglia organoids on day 14; n = 3 independent infections of 03SF microglia; one-tailed t test; ∗∗∗∗ p < 0.0001; data are presented as the mean ± SEM. (E) ELISA analysis of secreted p24 in culture supernatant from HIV-1 JRFL-infected or mock-infected microglia organoid day 14; n = 3 independent infections of 03SF microglia; one-tailed t test; ∗∗ p < 0.01; data are presented as the mean ± SEM. (F) RT-qPCR analysis of ISG15 , RSAD2 , IFITM3 , MX1 , IFI44 , and IFI27 mRNA from HIV-1 JRFL-infected or mock-infected microglia organoids on day 14; n = 3 independent infections of 03SF microglia; one-tailed t test; ∗ p < 0.05; data are presented as the mean ± SEM.

Article Snippet: The amount of viral p24 was determined using the HIV-1 Gag p24 DuoSet ELISA (R &D System, DY7360-05).

Techniques: Infection, Quantitative RT-PCR, One-tailed Test, Enzyme-linked Immunosorbent Assay

Journal: iScience

Article Title: Sustained type I interferon signaling after human immunodeficiency virus type 1 infection of human iPSC derived microglia and cerebral organoids

doi: 10.1016/j.isci.2024.109628

Figure Lengend Snippet:

Article Snippet: The amount of viral p24 was determined using the HIV-1 Gag p24 DuoSet ELISA (R &D System, DY7360-05).

Techniques: Virus, Recombinant, SYBR Green Assay, Enzyme-linked Immunosorbent Assay, RNA Sequencing, Software

Journal: Journal of Virology

Article Title: SARS-CoV-2 and SARS-CoV Spike-Mediated Cell-Cell Fusion Differ in Their Requirements for Receptor Expression and Proteolytic Activation

doi: 10.1128/JVI.00002-21

Figure Lengend Snippet: Requirements for the entry of SARS2-S-pseudotyped lentiviral particles differ from requirements for SARS2-S-mediated cell-cell fusion. (A) 293T cells transfected with an empty vector or ACE2/TMPRSS2, ACE2, or TMPRSS2 plasmid were preincubated with bromhexine, ambroxol, or camostat at the indicated concentration before the addition of lentiviral particles pseudotyped with SARS2-S. Forty-eight hours after transduction, the cells were analyzed via flow cytometry. Data show averaged percentages of GFP-positive cells, and error bars represent the standard deviations from one representative experiment performed in triplicate. (B) Micrographs of ACE2- and TMPRSS2-transfected cells that were infected with the respective lentiviral GFP-encoding pseudotype particles. (C) 293T cells transfected with ACE2/TMPRSS2 were preincubated with batimastat (10 μM), bromhexine (50 μM), ambroxol (50 μM), AEBSF (200 μM), camostat (50 μM), or batimastat (10 μM) in combination with camostat (50 μM) before the addition of lentiviral particles pseudotyped with the respective glycoprotein. Forty-eight hours after transduction, the cells were lysed and luciferase activity was determined. Data show fold changes over background (bald particles with solvent control), and error bars represent the standard deviations from three independent experiments, each performed in triplicate; raw values were log 10 transformed before analysis. (D) Western blot analysis of incorporation of the respective spike variants into lentiviral particles used in panel E and lysate control of transfected 293T cells used for the production of lentiviral particles. p24 and GAPDH served as loading controls. (E) 293T cells transfected with ACE2/TMPRSS2 were preincubated with batimastat (10 μM), E64-d (25 μM), camostat (50 μM), or batimastat (10 μM)/E64-d (25 μM) in combination with camostat (50 μM) before the addition of lentiviral particles pseudotyped with the respective glycoproteins. Forty-eight hours after transduction, the cells were lysed and luciferase activity was determined. Data show fold changes over background (bald particles with the solvent control), and error bars represent the standard deviations from two independent experiments, each performed in triplicate; raw values were log 10 transformed before analysis. Statistical significance in panels A, C, and E was determined by two-way ANOVA, and P values were corrected for multiple comparisons by Sidak’s method ( P > 0.05, ns; P ≤ 0.05, *; P ≤ 0.01, **; P ≤ 0.001, ***; P ≤ 0.0001, ****).

Article Snippet: Protein expression was analyzed by polyacrylamide gel electrophoresis on 8% to 16% precast gradient gels (Thermo) and Western blotting using antibodies to ACE2 (AF933; R&D Systems), the c-Myc epitope (clone 9E10; Santa Cruz Biotechnology), SARS spike (NB100-56578; Novus Biologicals), HIV-1 Gag p24 (clone 749140; R&D), and GAPDH (glyceraldehyde-3-phosphate dehydrogenase; GenScript) in NETT-G (150 mM NaCl, 5 mM EDTA, 50 mM Tris, 0.05% Triton X-100, 0.25% gelatin, pH 7.5) and donkey anti-mouse horseradish peroxidase (HRP)-coupled (Dianova), goat anti-rabbit HRP-coupled (Life Technologies), or rabbit anti-goat HRP-coupled (Proteintech) secondary antibody in 5% dry milk powder in PBS with 0.05% Tween 20.

Techniques: Transfection, Plasmid Preparation, Concentration Assay, Transduction, Flow Cytometry, Infection, Luciferase, Activity Assay, Solvent, Control, Transformation Assay, Western Blot