supt1 cd4 t cells  (Thermo Fisher)

Journal: PLoS ONE

Article Title: V3-Independent Competitive Resistance of a Dual-X4 HIV-1 to the CXCR4 Inhibitor AMD3100

doi: 10.1371/journal.pone.0089515

Figure Lengend Snippet: (A) Induction of AMD3100-resistant variants from HIV-1 JR-FLan/KI812.7 . Replication-competent HIV-1 JR-FLan/KI812.7 was passaged using SupT1/CCR5 cells in increasing concentrations of AMD3100 in the range of 20 nM to 4 µM. (B) Susceptibilities of AMD3100-selected variants to AMD3100 and MVC. TZM-bl cells were treated with various concentrations of AMD3100 or MVC, and infected with wild-type HIV-1 JR-FLan/KI812.7 , the virus passaged in the absence of AMD3100, or the selected virus in the presence of 4 µM AMD3100. Luciferase activities of TZM-bl cells were measured at 48 h post-infection. Data represent the extent of inhibition of replication relative to that in the absence of AMD3100 or MVC.

Article Snippet: The human CD4+ T cell line SupT1 was obtained from ATCC, and its derivative cell line SupT1/CCR5, which expressed high levels of CCR5, was established using a retroviral vector as described previously , , and maintained in RPMI 1640 (Sigma) medium supplemented with 10% FBS, 0.2 mg/mL G418, 100 U/mL penicillin, and 100 µg/mL streptomycin.

Techniques: Infection, Luciferase, Inhibition

Journal: PLoS ONE

Article Title: V3-Independent Competitive Resistance of a Dual-X4 HIV-1 to the CXCR4 Inhibitor AMD3100

doi: 10.1371/journal.pone.0089515

Figure Lengend Snippet: Amplified products from infected SupT1/CCR5 cells in the absence or presence of AMD3100 were cloned, and five to six clones from each sample were sequenced. The amino acid sequences of V2, C2, and C4 of the wild-type HIV-1 JR-FLan/KI812.7 are shown in the top line. In each set of clones, the deduced amino acid sequence was aligned by the single amino acid code. Identity with this sequence at individual amino acid positions is indicated by dots.

Article Snippet: The human CD4+ T cell line SupT1 was obtained from ATCC, and its derivative cell line SupT1/CCR5, which expressed high levels of CCR5, was established using a retroviral vector as described previously , , and maintained in RPMI 1640 (Sigma) medium supplemented with 10% FBS, 0.2 mg/mL G418, 100 U/mL penicillin, and 100 µg/mL streptomycin.

Techniques: Amplification, Infection, Clone Assay, Sequencing

Journal: PLoS ONE

Article Title: V3-Independent Competitive Resistance of a Dual-X4 HIV-1 to the CXCR4 Inhibitor AMD3100

doi: 10.1371/journal.pone.0089515

Figure Lengend Snippet: SupT1/CCR5 cells were infected with the same amount of replication-competent recombinant viruses carrying mutations (100 TCID 50 ) in the presence of various concentrations of AMD3100, and then cultured for 6 days. Cytopathic effects were determined by an MTT assay. Data are the means ± SD of triplicate experiments.

Article Snippet: The human CD4+ T cell line SupT1 was obtained from ATCC, and its derivative cell line SupT1/CCR5, which expressed high levels of CCR5, was established using a retroviral vector as described previously , , and maintained in RPMI 1640 (Sigma) medium supplemented with 10% FBS, 0.2 mg/mL G418, 100 U/mL penicillin, and 100 µg/mL streptomycin.

Techniques: Infection, Recombinant, Cell Culture, MTT Assay

Journal: bioRxiv

Article Title: High-resolution HIV-1 m 6 A epitranscriptome reveals splicing-dependent methylation clusters and unique 2-LTR transcript modifications

doi: 10.1101/2025.03.06.641803

Figure Lengend Snippet: (A) Outline of the infection and Nanopore sequencing protocols . SupT1 T cells or primary CD4 + T cells were infected with HIV NL4-3 pseudotyped with VSV-G to enhance viral infection. At 20 or 30 hours post-infection (pi) for HIV NL4-3 -infected SupT1 cells and at 48 h or 72 h pi for HIV NL4-3 -infected primary CD4 + T cells, we collected a fraction of the cell culture. The viability and percentages of HIV-Gag + and CD4 + cells were assessed by flow cytometry. During HIV infection, cells start expressing HIV-Gag protein and progressively lose their membrane expression of CD4 due to expression of the HIV accessory proteins Nef and Vpu, and HIV-Env. The cells were harvested and polyA RNA was purified and sequenced with Nanopore technology. Reads were mapped onto the HIV NL4-3 genome sequence. Created with BioRender. Moris, A. (2025) https://BioRender.com/o40u811 (B) Analysis of the infection rate of HIV NL4-3 -infected CD4 + SupT1 cells. After 20 h or 30 h, cells infected with HIV NL4-3 or mock-treated were labeled with a viability dye and stained with antibodies specific for CD4 and HIV-Gag. Left panels: the percentage viable cells. Middle and right panels: percentages of CD4 - HIV-Gag + and CD4 + HIV-Gag + cells among mock-treated and HIV NL4-3 -infected CD4 + SupT1 cells, respectively. (C) Analysis of the infection rate of HIV NL4-3 -infected primary CD4 + T cells. After 48 h or 72 h, cells from donors 864 and 880 were harvested and stained as in (B). Left panels: the percentage viable cells. Middle and right panels: percentages of CD4 - HIV-Gag + and CD4 + HIV-Gag + cells among mock-treated and HIV NL4-3 -infected primary CD4 + T cells, respectively, from donors 864 (top panels) and 880 (bottom panels). Numbers indicated the percentage of cells within the quadrant. MOCK: cells mock-treated without infection used as a negative control.

Article Snippet: 2 x 10 8 SupT1 CD4 + T cells were infected with 800 ng of VSV-G-HIV NL4-3 XCS HIV-Gagp24 for 3h in IMDM plus 10mM HEPES (Gibco/ThermoFisher Scientific) supplemented with 2 µg/ml of DEAE-dextran (Sigma).

Techniques: Infection, Nanopore Sequencing, Cell Culture, Flow Cytometry, Expressing, Membrane, Purification, Sequencing, Labeling, Staining, Negative Control

Journal: bioRxiv

Article Title: High-resolution HIV-1 m 6 A epitranscriptome reveals splicing-dependent methylation clusters and unique 2-LTR transcript modifications

doi: 10.1101/2025.03.06.641803

Figure Lengend Snippet: Detection of m 6 A sites in all HIV-1 transcripts. (A) The IGV screenshot shows the methylation rate measured in the cluster 1 region of the HIV-1 genome including sites A8436 to A8632. Line 1, methylation rate measured directly during the m 6 A base-call of SupT1_20h_pi rep1. Line 2, methylation rate measured directly for the in vitro sample in the same region. Line 3, Difference (line 1) - (line 2); red arrows indicate the eight sites detected; negative values are set to zero; sites with rates < 3% were not retained (black arrow). Vertical lines, positions of As in the five DRACH sequences of the region. (B) Methylation profile of all HIV-1 transcripts in the 3’ region (8360–9700). Closed red circles, SupT1_20h_pi rep1; empty red circles, SupT1_30h_pi rep1; closed green circles, SupT1_20h_pi rep2; empty green circles, SupT1_30h_pi rep2; closed black circles, CD4_48h_pi; empty black circles, CD4_72h_pi. Vertical lines, positions of As in the 33 DRACH sequences of the region.

Article Snippet: 2 x 10 8 SupT1 CD4 + T cells were infected with 800 ng of VSV-G-HIV NL4-3 XCS HIV-Gagp24 for 3h in IMDM plus 10mM HEPES (Gibco/ThermoFisher Scientific) supplemented with 2 µg/ml of DEAE-dextran (Sigma).

Techniques: Methylation, In Vitro

Journal: bioRxiv

Article Title: High-resolution HIV-1 m 6 A epitranscriptome reveals splicing-dependent methylation clusters and unique 2-LTR transcript modifications

doi: 10.1101/2025.03.06.641803

Figure Lengend Snippet: Methylation rates for the 14 m 6 A sites detected in SupT1_20h_pi rep1. (A) Unsupervised heatmap of the methylation rates of HIV-1 transcripts (Materials and Methods). Isoforms are clustered based on the methylation rates of the 14 m 6 A sites. The clusters essentially correspond to US, PS and CS transcripts. (B) Methylation rates of m 6 A sites in the CS, PS and US transcripts of the SupT1_20h_pi rep1 sample.

Article Snippet: 2 x 10 8 SupT1 CD4 + T cells were infected with 800 ng of VSV-G-HIV NL4-3 XCS HIV-Gagp24 for 3h in IMDM plus 10mM HEPES (Gibco/ThermoFisher Scientific) supplemented with 2 µg/ml of DEAE-dextran (Sigma).

Techniques: Methylation

Journal: bioRxiv

Article Title: High-resolution HIV-1 m 6 A epitranscriptome reveals splicing-dependent methylation clusters and unique 2-LTR transcript modifications

doi: 10.1101/2025.03.06.641803

Figure Lengend Snippet: m 6 A sites detected in splicing isoforms. (A) SupT1_20h_pi rep 1 sample; (B) SupT1_30h_pi rep1 sample; (C) SupT1_20h_pi rep2 sample; (D) SupT1_30h_pi rep2 sample; (E) CD4_48h_pi sample; (F) CD4_72h_pi sample. Only positions with p -values ≤ 1 x 10 -5 and a number of modified reads ≥ 30 are displayed.

Article Snippet: 2 x 10 8 SupT1 CD4 + T cells were infected with 800 ng of VSV-G-HIV NL4-3 XCS HIV-Gagp24 for 3h in IMDM plus 10mM HEPES (Gibco/ThermoFisher Scientific) supplemented with 2 µg/ml of DEAE-dextran (Sigma).

Techniques: Modification

Journal: bioRxiv

Article Title: High-resolution HIV-1 m 6 A epitranscriptome reveals splicing-dependent methylation clusters and unique 2-LTR transcript modifications

doi: 10.1101/2025.03.06.641803

Figure Lengend Snippet: Position and methylation rates of the four isoform-specific m 6 A sites. (A) The A5613, A5654 and A5703 sites are common to the Vif and Vpr isoforms; A5887 is common to the Vif, Vpr, Tat and SORF1 isoforms. (B) Methylation rates in SupT1_20h_pi rep1, calculated for the four sites in the CS (-intron 4, Vif1, Vpr1, Vpr2), PS (+ intron 4, Vif2, Vpr3, Vpr4) and US isoforms. (C) Methylation rates calculated for the A5887 site for the CS (Tat1, 2, 3, 4, SORF1), PS (Tat5) and US isoforms.

Article Snippet: 2 x 10 8 SupT1 CD4 + T cells were infected with 800 ng of VSV-G-HIV NL4-3 XCS HIV-Gagp24 for 3h in IMDM plus 10mM HEPES (Gibco/ThermoFisher Scientific) supplemented with 2 µg/ml of DEAE-dextran (Sigma).

Techniques: Methylation

Journal: bioRxiv

Article Title: High-resolution HIV-1 m 6 A epitranscriptome reveals splicing-dependent methylation clusters and unique 2-LTR transcript modifications

doi: 10.1101/2025.03.06.641803

Figure Lengend Snippet:

Article Snippet: 2 x 10 8 SupT1 CD4 + T cells were infected with 800 ng of VSV-G-HIV NL4-3 XCS HIV-Gagp24 for 3h in IMDM plus 10mM HEPES (Gibco/ThermoFisher Scientific) supplemented with 2 µg/ml of DEAE-dextran (Sigma).

Techniques: Methylation

Journal: bioRxiv

Article Title: High-resolution HIV-1 m 6 A epitranscriptome reveals splicing-dependent methylation clusters and unique 2-LTR transcript modifications

doi: 10.1101/2025.03.06.641803

Figure Lengend Snippet: Single-molecule analysis of methylated m 6 A sites. (A) Cluster 1. (B) Cluster 2. (C-E) Correlation between the number of methylated sites in cluster 1 and in cluster 2 of the same transcript from the SupT1_20h_pi rep1 sample. The intensity of the color on the heatmaps indicates the number of reads showing the indicated numbers of methylated As in cluster 1 (0 to 8) and in cluster 2 (0 to 3) (Materials and Methods).

Article Snippet: 2 x 10 8 SupT1 CD4 + T cells were infected with 800 ng of VSV-G-HIV NL4-3 XCS HIV-Gagp24 for 3h in IMDM plus 10mM HEPES (Gibco/ThermoFisher Scientific) supplemented with 2 µg/ml of DEAE-dextran (Sigma).

Techniques: Methylation

Journal: bioRxiv

Article Title: High-resolution HIV-1 m 6 A epitranscriptome reveals splicing-dependent methylation clusters and unique 2-LTR transcript modifications

doi: 10.1101/2025.03.06.641803

Figure Lengend Snippet: Single-molecule analysis of methylated m 6 A sites. (A) Cluster 1 and (B) cluster 2 of the indicated SupT1 samples. (C) Cluster 1 and (D) cluster 2 of the indicated CD4 samples.

Article Snippet: 2 x 10 8 SupT1 CD4 + T cells were infected with 800 ng of VSV-G-HIV NL4-3 XCS HIV-Gagp24 for 3h in IMDM plus 10mM HEPES (Gibco/ThermoFisher Scientific) supplemented with 2 µg/ml of DEAE-dextran (Sigma).

Techniques: Methylation

Journal: bioRxiv

Article Title: High-resolution HIV-1 m 6 A epitranscriptome reveals splicing-dependent methylation clusters and unique 2-LTR transcript modifications

doi: 10.1101/2025.03.06.641803

Figure Lengend Snippet: Transcription and methylation of the 2-LTR transcripts. (A) Diagram of the 2-LTR circles and of the transcripts joining the RU5 segment of the 3’ LTR to the U3R segment of the 5’ LTR. Several examples of insertion variants at the U5-U3 junction are shown. (B) Sequence of the 2-LTR transcripts. The successive transcribed regions — R, U5, U3, R — are indicated; m 6 A sites are shown in bold typeface and underlined and are named according to the position of the methylated A in the 2-LTR transcript; in brackets, the homologous position in the HIV-1 reference genome; for example, for 2L125(578), 125 is the position of the m 6 A in the 2-LTR transcript and (578) is the homologous position in the HIV-1 reference genome; * indicates that the sequence context of 2L183 in the 2-LTR transcript (AG A CT, see B) differs from that at the homologous position 9074 in the reference genome (GG A CT). (C) Correspondence table for a position in the 2-LTR transcript and the homologous position in the reference genome. (D) Sites detected in the 2-LTR transcripts of infected SupT1 and CD4 + T-cell samples (Materials and Methods). Large red circles, SupT1_20h_pi rep1; orange circles, SupT1_30h_pi rep1; blue circles, SupT1_20h_pi rep2; green circles, SupT1_30h_pi rep2; yellow circles, CD4_48h_pi; black circles, CD4_72h_pi. Vertical lines indicate the positions of the As in all the DRACH motifs of the transcript.

Article Snippet: 2 x 10 8 SupT1 CD4 + T cells were infected with 800 ng of VSV-G-HIV NL4-3 XCS HIV-Gagp24 for 3h in IMDM plus 10mM HEPES (Gibco/ThermoFisher Scientific) supplemented with 2 µg/ml of DEAE-dextran (Sigma).

Techniques: Methylation, Sequencing, Infection

Journal: bioRxiv

Article Title: Unveiling novel conserved HIV-1 open reading frames encoding T cell antigens using ribosome profiling

doi: 10.1101/2024.11.12.623167

Figure Lengend Snippet: (a) Distribution of ribosome protected fragments (RPFs) across HIV-1 genome obtained from HIV NL4-3 -infected SupT1 cells. The number of RPFs (Nb of reads) are represented according to the position in HIV NL4-3 genome. For clarity, the numbers of RPFs are truncated above 5000 reads. To illustrate the translation signal in the UTR duplicated regions, multimapped RPF are shown. RPFs translated in frames 0, +1, and +2 are represented in green, red, and blue, respectively. HIV genomic RNA is represented below; CDSs are indicated and colored accordingly to their reading frames from the first position of the genome. Note that the classical reference strain, HIV HxB2, owns an extra T (position 5772 within the vpr gene) as compared to most clade B strains. As a direct consequence in the HIV NL4-3 genome, env CDS is not in the same reading frame as pol and vpr CDSs. The CDSs from tat, rev, vpu and nef frames are also in a different frame than in the HIV HxB2 genome. The positioning of ARFs (purple lines) are indicated in the lower RNA genome representation. The selection criteria were: 1) not corresponding to CDS sequences; 2) aa length ≥ 10; 3) translated between 2 stops codons and 4) reads per codon ≥ 8. (b) Relative expression of HIV CDSs. Relative expressions of HIV NL4-3 CDSs were obtained by dividing the total read counts of each RPF by its length establishing a read/nucleotide value. As in (A), the color codes indicate the frames of in which the ARFs are localized (green, 0; red, +1; blue, +2). (c) Ribosome densities reveal novel viral coding regions. The number of RPFs (Nb of reads) are represented (y-axis) according to the position in HIV NL4-3 genome (x-axis). The color codes indicate the frames of the ARF and the relative CDS (green, 0; red, +1; blue, +2). Filled and open rectangles indicate the relative CDS and the ARF, respectively. ARFs within gag (top left), vif (top right), env (bottom left), and nef (bottom right) CDSs. Putative start and stop codons are labelled according to their reading frames with triangles and crosses, respectively. (d) Pie chart of the genomic distribution of the 98 identified ARFs according to the overlapping CDS. ARFs that overlap two CDS sequences are group into segments labelled with the name of the 2 CDSs. The number of ARFs is indicated below the name.

Article Snippet: 1 x 10 SupT1 CD4 + T cells were infected with 4 055 ng of VSV-G-HIV NL4-3 XCS HIV-Gagp24 for 3h in IMDM plus 10mM HEPES (Gibco/ThermoFisher Scientific) supplemented with 2 µg/ml of DEAE-dextran (Sigma).

Techniques: Infection, Selection, Expressing

Journal: bioRxiv

Article Title: Unveiling novel conserved HIV-1 open reading frames encoding T cell antigens using ribosome profiling

doi: 10.1101/2024.11.12.623167

Figure Lengend Snippet:

Article Snippet: 1 x 10 SupT1 CD4 + T cells were infected with 4 055 ng of VSV-G-HIV NL4-3 XCS HIV-Gagp24 for 3h in IMDM plus 10mM HEPES (Gibco/ThermoFisher Scientific) supplemented with 2 µg/ml of DEAE-dextran (Sigma).

Techniques: Infection, Selection, Sequencing

Journal: bioRxiv

Article Title: Unveiling novel conserved HIV-1 open reading frames encoding T cell antigens using ribosome profiling

doi: 10.1101/2024.11.12.623167

Figure Lengend Snippet: (a) Representative gating scheme for the identification of ARFP-specific T cell responses. Upper panel, gatings for G02-ARFP-specific CD4 + (left) and CD8 + (right) T cell responses are shown for the EC-3 HIV-infected individual. Lower panel, gatings for Env01-specific T cell responses as for G02. Env01 epitope is a known immunodominant peptide from HIV-Env. CD8 + CD4 - T cell populations were pre-gated on CD8 + CD3 + T-cells, lived cells and doublets were excluded. Gates for each cytokine/chemokine were set based on the negative controls (cells loaded with DMSO containing medium, NS: Not stimulated) NS_ARF and NS_CDS for the assessment of G02-ARFP- and Env01-specific T cell responses, respectively. A figure exemplifying the gating strategy is provided in the Supplementary Fig. 13 (b) Heatmap of ARFP recognized by CD4 + or CD8 + T cells from HIV-infected donors summarizing the ICS assays. NA = Not applicable (i.e, not tested), NS = Not significant. (c) Percentage of CD4 + or CD8 + T cell responses among ARFP-specific T cell responses after in vitro stimulation with ARFP at the cohort level. Proportion of ARFP- and CDS-specific CD4 + CD3 + (d) or CD8 + CD3 + (e) T-cells secreting 1 to 5 cytokines simultaneously. The numbers on the top indicate the percentage of polyfunctional T cells secreting at least 3 cytokines. ARFP-specific T cell responses from all tested donors are combined. (f) Comparison of the percentage of polyfunctional T cells secreting at least 3 cytokines among ARFP- and CDS-specific CD4 + CD3 + T cells in the EC and ART patient groups. (g) same as (f) among ARFP- and CDS-specific CD8 + CD3 + T cells in EC group. Each dot represents one peptide-specific T cell response. The lines correspond to the median responses. Mann-Whitney test with Dunn’s comparison was applied. ns: not significant. From (d), data for YSL-I- and YSL-II-specific T cell responses are integrated into the graphs.

Article Snippet: 1 x 10 SupT1 CD4 + T cells were infected with 4 055 ng of VSV-G-HIV NL4-3 XCS HIV-Gagp24 for 3h in IMDM plus 10mM HEPES (Gibco/ThermoFisher Scientific) supplemented with 2 µg/ml of DEAE-dextran (Sigma).

Techniques: Infection, In Vitro, Comparison, MANN-WHITNEY

Journal: bioRxiv

Article Title: Unveiling novel conserved HIV-1 open reading frames encoding T cell antigens using ribosome profiling

doi: 10.1101/2024.11.12.623167

Figure Lengend Snippet: (a) Identification of CDS and ARFP from HIV NL4-3 -infected primary CD4 + T cells. The sequences of identified HIV-derived HLA class I ligands and their characteristics are presented. (b) Detection of T cell responses specific to YSL ARF-derived peptide in PBMCs of HIV-1 infected donors. The IFNγ responses of donors EC-1 and EC3 who responded to the YSL-peptide stimulation, on day 12, are presented. Cells loaded with DMSO-containing medium, used as negative controls, (POOL (-)) are indicated by a white bar for each donor. POOL (+), YSL-I, and YSL-II correspond to cells loaded with a pool of peptides containing YSL-I and YSL-II peptides, or only with YSL-I and YSL-II peptides, respectively. A response was considered positive when the number of rough IFNγ + spots per well was >20 and 2 times higher than the negative control (POOL (-) white bar, cells loaded with DMSO-containing medium). SAT: saturated well for which the IFNγ signal was so high that it could not be quantified. (c) Functional characteristics of YSL-specific CD4 + T cell response from EC-3 donor . Following a 12-day in vitro expansion, intracellular staining was performed for MIP-1β, IFNγ, IL-2, CD107a, and TNF after stimulation with YSL-I (top dotplot panel) or an Env-derived peptide ( MHC-II_env01, bottom dotplot panel) used as positive control. Cells were gated on the CD4 + CD3 + population and each of the five individual gates for each cytokine/chemokine was set based on the negative controls (NS_, cells loaded with DMSO-containing medium). CD4 + CD3 + cells were pre-gated on lived cells and doublets were excluded. Right panel, the left pie charts illustrate the percentage of activated (yellow) and non-activated (blue) T cells following YSL- or Env-peptide stimulation, upper and lower lines, respectively. The polyfunctional profile of activated YSL- or Env-specific T cells are shown in the right pie charts. Boolean analysis was conducted to determine whether T cells secreted 1, 2, 3, 4 or 5 cytokines simultaneously (red, green, purple, blue, and orange, respectively). Numbers indicate the percentage of activated cells with one to five cytokines secreted simultaneously. Polyfunctional profiles of CD4 + T cell responses against YSL-I (d) and YSL-II (e) peptides. The left pie charts show the percentage of activated (yellow) and non-activated (blue) T cells following peptide stimulation. The polyfunctionality of activated T cells, for all responding donors, is shown as pie charts on the right. The donor names are indicated on the top. Boolean analysis was conducted to determine whether T cells secreted 1, 2, 3, 4, or 5 cytokines simultaneously (red, green, purple, blue, and orange, respectively). Numbers indicate the percentage of activated cells with one to five cytokines secreted simultaneously (f) Heatmap summarizing YSL-I and YSL-II peptide-specific CD4 + T cell responses in all tested HIV-infected donors. NA = Not applicable, NS = tested but Not significant.

Article Snippet: 1 x 10 SupT1 CD4 + T cells were infected with 4 055 ng of VSV-G-HIV NL4-3 XCS HIV-Gagp24 for 3h in IMDM plus 10mM HEPES (Gibco/ThermoFisher Scientific) supplemented with 2 µg/ml of DEAE-dextran (Sigma).

Techniques: Infection, Derivative Assay, Negative Control, Functional Assay, In Vitro, Staining, Positive Control

Journal: PLoS ONE

Article Title: The Lantibiotic Peptide Labyrinthopeptin A1 Demonstrates Broad Anti-HIV and Anti-HSV Activity with Potential for Microbicidal Applications

doi: 10.1371/journal.pone.0064010

Figure Lengend Snippet: (A) Light microscopical pictures of (a) persistently HIV-infected HUT-78/IIIB cells, (b) non-infected CD4 + target T cells, (c) giant cell formations after coculture of HUT-78/IIIB and SupT1 cells, (d) coculture of HUT-78/IIIB and SupT1 cells in the presence of 24 µM LabyA1, (e) 4.8 µM LabyA1 and (f) 0.96 µM LabyA1. One representative experiment is shown. Magnification x10/0.25. (B) Percentage of living SupT1 T cells after 24 h of coculture with HUT-78/IIIB cells in the presence of LabyA1. To discriminate SupT1 T cells from HUT-78/IIIB cells, the expression of the CD28 marker was measured by flow cytometry. The mean ± SEM out of 4 independent experiments is shown.

Article Snippet: The depletion of the target CD4 + SupT1 T cells in the cocultivation assays was measured using PE-conjugated anti-CD28 (BD Biosciences) .

Techniques: Infection, Expressing, Marker, Flow Cytometry

Journal: PLoS ONE

Article Title: The Lantibiotic Peptide Labyrinthopeptin A1 Demonstrates Broad Anti-HIV and Anti-HSV Activity with Potential for Microbicidal Applications

doi: 10.1371/journal.pone.0064010

Figure Lengend Snippet: (A) CD4 + SupT1 cells were incubated with 3 different anti-CD4 mAbs (RPA-T4, MT441 and OKT-4), in the presence of 9.6 µM or 1.9 µM of LabyA1 and antibody binding was measured by flow cytometry. Data represent the mean-values ± SEM of the percentage of anti-CD4 mAb binding of 2–3 independent experiments. (B) Binding of HIV-1 NL4.3, measured by the anti-gp120 (9205) mAb, to CD4 + SupT1 cells in the presence of LabyA1, sCD4 and AMD3100. MFI-values of the background fluorescence and virus binding are shown in green and grey histograms, respectively. One representative experiment out of 3 is shown. (C) Interaction with the CXCR4 receptor was measured by flow cytometry after incubation of SupT1 T cells with the anti-CXCR4mAb (12G5) in the presence of 9.6 µM and 1.9 µM of LabyA1. AMD3100 was included as reference compound. Data represent mean-values ± SEM ( n = 3) of the percentage of CXCR4 mAb binding according to the untreated control. * p <0.05, according to unpaired T-test. (D) Intracellular calcium mobilization in U87.CD4.CCR5 cells (upper panel) and in U87.CD4.CXCR4 cells (lower panel). Fluo-3 loaded cells pre-incubated in the absence or presence of 9.6 µM LabyA1 were stimulated with 10 ng/ml LD78β (upper panel) or 100 ng/ml SDF-1α (lower panel) or with 9.6 µM LabyA1 and the fluorescence change was monitored. One representative experiment out of 3 is shown. (E) MT-4 cells were pre-incubated with LabyA1, AMD3100 or T20, extensively washed and infected with HIV-1 NL4.3. Viral replication was measured 5 days post-infection. Mean CPE values ± SEM up to 5 experiments are shown. Statistical * p <0.05, according to unpaired T-test.

Article Snippet: The depletion of the target CD4 + SupT1 T cells in the cocultivation assays was measured using PE-conjugated anti-CD28 (BD Biosciences) .

Techniques: Incubation, Binding Assay, Flow Cytometry, Fluorescence, Infection