MOESM5 of The role of antigen presenting cells in the induction of HIV-1 latency in resting CD4+ T-cells

Additional file 5: Table S2. Comparison of gene expression between latency inducing and non-inducing antigen presenting cell subpopulations using microarray. Using the bioinformatics databases DAVID, GeneCards and GeneCodis, gene expression compartment and function was determined. Genes expressed on the antigen presenting cell (APC)-surface with the ability to signal to T-cells were shortlisted

MOESM1 of HIV latency reversing agents act through Tat post translational modifications

Additional file 1: Figure S1. Cellular toxicity of LRAs. The CellTiter 96 Aqueous One Solution Cell Proliferation MTS assay was used to measure the toxicity of a panel of LRAs on HEK293T cells over a range of concentrations (31.25 to 1000 nM) for 48 h. VOR = vorinostat; PAN = panobinostat; CTN = chaetocin; DIS = disulfiram. The lines represent the mean + SD (n = 2

MOESM5 of HIV latency reversing agents act through Tat post translational modifications

Additional file 5: Table S1. Oligonucleotides used in this study fo

MOESM3 of HIV latency reversing agents act through Tat post translational modifications

Additional file 3: Figure S3. Cellular and HIV RNA levels following JQ1 treatment. A. Absolute quantification of RPP30, IPO8 and TBP cellular mRNAs (copies/μl) were performed using total RNAs derived from transfected HEK293T cells with the pLTR.gp140/EGFP.Rev∆38/DsRed splicing reporter in the absence and presence of 100 ng of pTat101 (AD8)-Flag expression plasmid and treated with JQ1 (1 μM) or DMSO diluent control. B. HIV unspliced (US), spliced (D4-A7) and all viral RNA expression levels (copies/ul) were quantified by droplet digital PCR (ddPCR) and normalized over the 3 reference genes. Comparisons of each condition to DMSO were made using a paired T test. Only statistically significant comparisons are shown *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. The black lines represent the mean ± SEM (n = 4

MOESM2 of HIV latency reversing agents act through Tat post translational modifications

Additional file 2: Figure S2. JQ1 increases EGFP and DsRed expression from an LTR-driven splicing reporter in the absence and presence of Tat. HEK293T cells were transfected with the pLTR.gp140/EGFP.Rev∆38/DsRed splicing reporter in the absence or presence of 100 ng of pTat101 (AD8)-Flag expression plasmid and then treated for 24 h with JQ1 (1 μM) or DMSO diluent control. Cells were harvested and portion analysed for either the percentage of cells expressing EGFP (unspliced, A.) or DsRed (spliced, B.) using flow cytometry, or HIV unspliced (US), spliced (D4-A7) and all viral RNA expression levels (copies/ul) by droplet digital PCR (ddPCR) (Fig. 3). The fold-change (FC) over DMSO of Live+ EGFP+ (A.), Live + DsRed + (B.) and percentage of spliced product DsRed/(DsRed + EGFP) (C.) were determined. Comparisons of each condition to DMSO were made using a paired T test. Only statistically significant comparisons are shown **p < 0.01; ***p < 0.001; ****p < 0.0001. The black lines represent the mean ± SEM (n = 4

MOESM4 of HIV integration and the establishment of latency in CCL19-treated resting CD4+ T cells require activation of NF-ÎşB

Additional file 4: Figure S4. Integration site selection and gene activation in chemokine treated cells. A, Gene expression was determined by Illumina bead array in unactivated, CCL19-treated or PHA-IL2 activated CD4+ T cells after 6 or 72 h. The ratio of expression of genes at the sites of integration was determined in each in vitro condition. B, Expression of individual genes at the site of HIV integration in CCL19-treated resting CD4+ T cells (x-axis) compared to unactivated (y-axis; upper panel) or PHA-IL2 activated CD4+ T cells (y-axis; lower panel). C, The distance of integration sites to specific genomic elements including LINE, H4K20me3 and H4R3me following HIV infection of unactivated, CCL19-treated and PHA-IL2 activated CD4+ T cells, or CD4+ T cells from HIV-infected patients on cART or randomly selected sites. Log distance is shown as box plots (median and quartiles) with violin plot of the kernel distribution. The means are shown as a red horizontal line

Graphic summary of the ability of each compound to activate HIV within each cell model: (A) primary CD4 T cell models and patient cell outgrowth assay (QVOA), and (B) J-Lat T cell line clones.

<p>Each compound and concentration tested is listed on the X-axis. In the primary CD4 cell models, each compound was tested using cells from 2, 3 or 4 different donors and in duplicate or triplicate with cells from each donor (See <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003834#s4" target="_blank">Methods</a> Section for details). For the QVOA, results from the limiting dilution cultures from 3 patients were pooled to calculate one common IUPM (infectious units per million cells) value which was then normalized to that obtained with PHA. With the J-Lat clones, experiments were performed in triplicate. Asterisks represent “not done”.</p

List of stimuli used in this study and their corresponding signaling pathways.

<p>List of stimuli used in this study and their corresponding signaling pathways.</p

Heatmap visualization of the ability of each compound to activate HIV within each model when excluding (A) and including (B) data from the QVOA model.

<p>A reduced set of compounds was analyzed in (<b>B</b>) since not every compound was run at every concentration in the QVOA. The clustergram at the left of each heatmap reflects the relationships between compounds based on their ability to activate HIV across compounds. Since cells in all models responded to PHA with high strength, ranking was normalized within each model to the response to PHA at 10 µg/mL and, therefore, all models display in the heatmap the same relative responsiveness to this treatment. The clustergram at the top of each heatmap reflects the relationship between each model based on their response to compounds. Clustergrams were created by calculating Euclidean distances and then clustering distances using the average linkage method. The numbers at the nodes of clusters are AU p-values where 95% represents a <i>p</i>-value cut-off of 0.05 and only values 95% or greater are depicted. Red cells in the heatmaps reflect HIV activation whereas blue or blank cells indicate that the compound did not effectively activate HIV.</p

Discovery-Based Science Education: Functional Genomic Dissection in Drosophila by Undergraduate Researchers

Discovery-Based Science Education: Functional Genomic Dissection in Drosophila by Undergraduate Researcher