easy sep human t cell enrichment kit  (STEMCELL Technologies Inc)

Journal: bioRxiv

Article Title: Histone deacetylase inhibitors butyrate and bufexamac inhibit de novo HIV-1 infection in CD4 T-cells

doi: 10.1101/2020.04.29.067884

Figure Lengend Snippet: A) CD4 + T-cells were isolated from blood of healthy donors using RosetteSep™ technology. Subsequently, purity of isolated cells was analyzed by fluorescence-based flow cytometry using anti-CD3 / -CD4 staining. FACS plots display the gating strategy for living cells (lymphocytes; left), singlets (middle), and enriched CD4 + T-cells (right). Numbers display the percentage of cells within the respective gate. B) Representative FACS plots of CD4 + T-cell isolation based on the gating strategy in A from four different donors, which were used later in four-donor-pools. C) Schematic illustration of T-cell activation. Isolated CD4 + T-cells (resting) were cultivated in the presence of PHA and IL-2 for 5-8 days to gain activated CD4 + T-cells. D) Representative FACS plots of the isolated cells from B after activation. CD69 was used as activation marker.

Article Snippet: For isolation of CD4 + T-cells, blood cells were diluted with PBS (Gibco) and T-cells were isolated via the Easy-Sep™ Rosette Human CD4 + T-cell enrichment kit (Stemcell Technologies, Canada) according to manufacturer’s protocols.

Techniques: Isolation, Fluorescence, Flow Cytometry, Staining, Cell Isolation, Activation Assay, Marker

Journal: bioRxiv

Article Title: Histone deacetylase inhibitors butyrate and bufexamac inhibit de novo HIV-1 infection in CD4 T-cells

doi: 10.1101/2020.04.29.067884

Figure Lengend Snippet: Activated CD4 + T-cells were cultivated in the presence of different concentrations of the respective HDACIs for 72h. Afterwards, viability of cells was determined using a luminescence-based viability assay. Besides individual data points the fitting curve of all measurements, R 2 value, Hill slope (Hs), and lethal concentration of 20% (LC 20 ) is displayed. Data are derived from three independent experiments each with 2 independent four-donor pools. Error bars show standard deviation (SD). For Romidepsin the mean of three independent experiments as well as concentration-dependent viability curves of 4 individual donors are shown (upper left graph).

Article Snippet: For isolation of CD4 + T-cells, blood cells were diluted with PBS (Gibco) and T-cells were isolated via the Easy-Sep™ Rosette Human CD4 + T-cell enrichment kit (Stemcell Technologies, Canada) according to manufacturer’s protocols.

Techniques: Viability Assay, Concentration Assay, Derivative Assay, Standard Deviation

Journal: bioRxiv

Article Title: Histone deacetylase inhibitors butyrate and bufexamac inhibit de novo HIV-1 infection in CD4 T-cells

doi: 10.1101/2020.04.29.067884

Figure Lengend Snippet: A) Experimental design. Isolated, primary CD4 + T-cells were activated by PHA and IL-2 treatment for 5-8 days. Derived activated T-cells were treated with HDACIs for 24 h and subsequently infected with HIV-1 NL4-3 . 48 h post infection, cells were stained for the viral p24 capsid protein and analyzed by flow cytometry. B) Representative FACS histogram plot showing the increase of p24 capsid protein in infected cells (green). Uninfected cells were used as control (grey) C) Box-Whisker plots representing the HIV-1 NL4-3 infection levels in CD4 + T-cells in dependency of the respective HDACI treatment. All data were normalized to the median of vehicle control samples. Boxes represent the lower and upper quartiles, whiskers show the minimum and maximal values, and the line inside the box indicates the median. Individual data points are presented as red dots. Sample size: n ≥ 10. Given p-values show statistically difference (ANOVA-based Dunnetts’ multiple comparison test) between vehicle control and sodium butyrate and bufexamac treated cells. D) Graph similar to C except infection with R5-tropic HIV-1 T/F CH058 virus. Sample size: n = 9.

Article Snippet: For isolation of CD4 + T-cells, blood cells were diluted with PBS (Gibco) and T-cells were isolated via the Easy-Sep™ Rosette Human CD4 + T-cell enrichment kit (Stemcell Technologies, Canada) according to manufacturer’s protocols.

Techniques: Isolation, Derivative Assay, Infection, Staining, Flow Cytometry, Whisker Assay

Journal: bioRxiv

Article Title: Histone deacetylase inhibitors butyrate and bufexamac inhibit de novo HIV-1 infection in CD4 T-cells

doi: 10.1101/2020.04.29.067884

Figure Lengend Snippet: A and B) Volcano plots of identified transcripts showing the log 2 fold change of abundance between inhibitor (A: Sb, B: Bu) and vehicle control and the associated q-value for each transcript. Colour code: Red indicates significantly upregulated transcripts (FDR ≤ 001 and log 2 fold change ≥ 1), Blue indicates significantly downregulated transcripts (FDR ≤ 001 and log 2 fold change ≤ −1), and Grey indicates significantly differential expressed genes, which are not up- or down-regulated (FDR ≤ 001). C). GO-term and pathway enrichment analysis of significantly down-regulated genes in Sb- (left) and Bu-treated cells (right) based on the g:profiler analysis tool. Representative terms are shown. The graphs display q-values and amount of regulated genes for each indicated GO-term and KEGG pathway. See also Figure S6C. D) Cell cycle analysis. Jurkat-E6 cells were treated with Sb or Bu for 48 h. Afterwards, cells were fixed, stained with propidium iodide (PI) and analyzed by flow cytometry. Cell cycle phases were determined using FlowJo software. Histogramms on the left show representative measurements. Donut charts on the right display the amount of cells in the different cell cycle phases as derived from 3 independent experiments. E) Determination of 2-LTR circles. Graph displays the percentage of HIV-1-based 2-LTR circles measured in compound-treated CD4 + T-cells. Romidepsin (Ro) was used as internal control. Data are derived from 3 independent experiments and are normalized to vehicle-treated control cells. Error bars display SD. Statistics was performed using the ANOVA-based Tukeys’ multiple comparison test.

Article Snippet: For isolation of CD4 + T-cells, blood cells were diluted with PBS (Gibco) and T-cells were isolated via the Easy-Sep™ Rosette Human CD4 + T-cell enrichment kit (Stemcell Technologies, Canada) according to manufacturer’s protocols.

Techniques: Cell Cycle Assay, Staining, Flow Cytometry, Software, Derivative Assay

Journal: bioRxiv

Article Title: Histone deacetylase inhibitors butyrate and bufexamac inhibit de novo HIV-1 infection in CD4 T-cells

doi: 10.1101/2020.04.29.067884

Figure Lengend Snippet: A) Correlation plot of RNA-Seq data from Sb- and Bu-treated cells. Only transcripts are shown, which could be assigned to an ENSEMBL ID. DEGs with log 2 fold change > −1 and < 1 are depicted in grey. Regulated DEGs (log 2 fold change ≥ 1 or ≤ −1) are indicated as follows: upregulated in only one condition (orange), upregulated in both conditions (red), downregulated in only one condition (light blue), downregulated in both conditions (dark blue), and contrary regulated (magenta). n = number of DEGs belonging to the respective group. B) Heatmaps showing the 20 most up- or down-regulated genes in Sb-treated (left) and Bu-treated (right) cells. Colour intensity shows row z-score of variance stabilized transformation (VST) values. Top 20 genes that have been identified in both treatment conditions are indicated with red letters. C) GO-term-analysis of up-regulated genes. Left: Sb-treatment, Right: Bu-treatment. Representative terms are shown. The graphs display q-value and amount of regulated genes for each indicated GO-term. See also . D) Viral particle fusion assay. Box-Whisker plot displays the percentage of fusion of viral particles with the target cell. Datasets were normalized to untreated control cells and were derived from three independent experiments. Boxes show the lower and upper quartiles, whiskers show the minimum and maximal values, and the line inside the box indicates the median. Statistics was performed using one-way ANOVA-based Dunnetts’ multiple comparison test. Sb: Sodium Butyrate, Bu: Bufexamac, Ro: Romidepsin. E) RT activity assay. Graph displays RT activity in dependency of HDACI treatment as analyzed in HIV-1 infected CD4 + T-cells. Data were derived from four independent experiments; error bars show SD.

Article Snippet: For isolation of CD4 + T-cells, blood cells were diluted with PBS (Gibco) and T-cells were isolated via the Easy-Sep™ Rosette Human CD4 + T-cell enrichment kit (Stemcell Technologies, Canada) according to manufacturer’s protocols.

Techniques: RNA Sequencing Assay, Transformation Assay, Single Vesicle Fusion Assay, Whisker Assay, Derivative Assay, RT Activity Assay, Activity Assay, Infection

Journal: bioRxiv

Article Title: Histone deacetylase inhibitors butyrate and bufexamac inhibit de novo HIV-1 infection in CD4 T-cells

doi: 10.1101/2020.04.29.067884

Figure Lengend Snippet: CD4 + T-cells were treated with bufexamac in presence of increasing concentrations of iron (FeCl 3 ). 24 h post treatment, cells were infected with HIV-1 NL4-3 and further cultured for another 48 h. Subsequently, cells were analyzed for p24 via flow cytometry. Untreated cells were used for control.

Article Snippet: For isolation of CD4 + T-cells, blood cells were diluted with PBS (Gibco) and T-cells were isolated via the Easy-Sep™ Rosette Human CD4 + T-cell enrichment kit (Stemcell Technologies, Canada) according to manufacturer’s protocols.

Techniques: Infection, Cell Culture, Flow Cytometry

Journal: bioRxiv

Article Title: Histone deacetylase inhibitors butyrate and bufexamac inhibit de novo HIV-1 infection in CD4 T-cells

doi: 10.1101/2020.04.29.067884

Figure Lengend Snippet: A) Schematic illustration of Sb-sample preparation. Cells were challenged for 48 h, lyzed, and subsequently proteins were digested by trypsin. Derived peptides were purified, labeled with isobaric TMT-tags, mixed and analyzed by LC-MS/MS. B) Dot plot displays all identified proteins and the associated log 2 fold change upon Sb-treament in comparison to control cells. Colored areas represent cut-offs for up-regulation (red; ≥ 2 x SD), down-regulation (blue; ≤ 2 x SD), and no regulation (≤ 2x SD ≥). Numbers (n) of regulated proteins are given on the upper (up-regulated) and lower (down-regulated) right side of the graph. Most changed proteins are indicated by protein name. C) Dot plot displays identified proteins in Bu-treated cells. Data were derived from and were reanalyzed according to similar thresholds as in B. D) Functional interaction network of regulated proteins in Sb-treated cells. Network analysis was performed using STRING database and networks were visualized via cytoscape. The color code displays the measured log 2 fold changes. Connected proteins within the network, which were down-regulated at protein but not at mRNA level are highlighted (bold black lines). E) Immunoblot of direct cell lysates from treated and HIV-1 infected CD4 + T-cells, respectively, as well as of co-immunoprecipitated samples, showing EP300 levels (left). Vinculin served as loading control. Graph on the right shows relative EP300 activity of enriched EP300 (IP samples). Boxes show the lower and upper quartiles, whiskers show the minimum and maximal values, and the line inside the box indicates the median. Statistics was performed using the ANOVA-based Tukeys’ multiple comparison test. F) EP300-associated HIV-1 infection levels. Bars show HIV-1 NL4-3 infection levels of CD4 + T-cells treated either with sodium butyrate or the EP300 inhibitor anarcadic acid (10µM). Data are derived from four experiments and error bars show SD. G) Immunoblots of intracellular EP300 levels in control and Sb-treated cells in presence or absence of proteasome inhibitor MG-132.

Article Snippet: For isolation of CD4 + T-cells, blood cells were diluted with PBS (Gibco) and T-cells were isolated via the Easy-Sep™ Rosette Human CD4 + T-cell enrichment kit (Stemcell Technologies, Canada) according to manufacturer’s protocols.

Techniques: Sample Prep, Derivative Assay, Purification, Labeling, Liquid Chromatography with Mass Spectroscopy, Functional Assay, Western Blot, Infection, Immunoprecipitation, Activity Assay