robosep negative selection human cd4 + t cell enrichment kit  (STEMCELL Technologies Inc)

Journal: EMBO Molecular Medicine

Article Title: Glycolysis downregulation is a hallmark of HIV‐1 latency and sensitizes infected cells to oxidative stress

doi: 10.15252/emmm.202013901

Figure Lengend Snippet: A–C Primary CD4 + T cells were activated with α‐CD3‐CD28 beads and infected with HIV‐1 pNL4‐3 or mock‐infected. Cells were cultured for 2 weeks post‐infection (p.i.) to model different infection stages (days 3–9 p.i., i.e., productive infection; day 14 p.i., i.e., latent infection) and subjected to microarray (A, B; n = 2) or RNA‐Seq (C; n = 3) analysis. (A) Gene set enrichment analysis (GSEA) of the expression of the glycolytic pathway (HUMAN‐GLYCOLYSIS) in mock‐infected or HIV‐1‐infected cells. (B,C) Heatmaps of the relative expression of glycolytic enzymes upon HIV‐1 infection. Data are expressed as Log 2 fold change in HIV‐1‐infected vs mock‐infected cells. For microarray data (B), expression values of infected and mock‐infected cells at different time points were pooled. For RNA‐Seq data (C), expression values in infected cells were normalized using the corresponding time point in mock‐infected cells. Adjusted P ‐values (q values) to account for multiple testing were calculated by Significance Analysis of Microarrays [SAM (Tusher et al , )] and Deseq2 for RNA‐Seq data (Love et al , ). D–F scRNA‐Seq of the expression of the entire glycolytic pathway or of glucose phosphate isomerase only ( GPI ) in primary CD4 + T cells infected in vitro (D,E) or CD4 + T cells of PLWH (F). In panels (D, E), cells were infected with VSVG‐HIV‐1‐GFP and sorted for viral expression as detailed in Golumbeanu et al . Following latency establishment, cells were left untreated or HIV‐1 expression was reactivated through suberoylanilide hydroxamic acid (SAHA) or α‐CD3‐CD28 engagement. Clusters 1 and 2 were identified by principal component analysis as described in Golumbeanu et al . In panel (F), CD4 + T cells were isolated from total blood of PLWH under ART as described in Cohn et al . Viral expression was reactivated by treatment with phytohemagglutinin (PHA), and cells were sorted using antibodies against Env and Gag. Sorted cells were then subjected to scRNA‐Seq analysis. The expression level of the HUMAN‐GLYCOLYSIS pathway in (D) was calculated as the average expression of genes comprising the gene list; expression levels in clusters 1 and 2 were compared using Wilcoxon rank‐sum test. For panels (E, F), significance of GPI differential expression level between clusters (E) or between control and Env + Gag + conditions (F) was assessed by the Wilcoxon rank‐sum test encoded in FindMarkers Seurat R function. ** P < 0.01, *** P < 0.001; *** P < 0.0001. For panels (D, E) n = 1 donor, 43 cells (untreated), 90 cells (SAHA), 91 cells (TCR), for panel (F) n = 3 donors, 109 cells (control) and 85 cells (Gag + Env + ).

Article Snippet: Briefly, naive CD4 + T cells were isolated using a RoboSep CD4 + Naïve T‐cell negative selection kit (STEMCELL Technologies Inc., Vancouver, British Columbia, Canada), and 2 × 10 6 cells were resuspended in 10 ml RPMI medium and stimulated with 10 μg/ml concanavalin A (ConA) (EMD Millipore, Billerica, MA, USA) in the presence of subset‐specific cytokines.

Techniques: Infection, Cell Culture, Microarray, RNA Sequencing, Expressing, In Vitro, Isolation, Quantitative Proteomics, Control

Journal: EMBO Molecular Medicine

Article Title: Glycolysis downregulation is a hallmark of HIV‐1 latency and sensitizes infected cells to oxidative stress

doi: 10.15252/emmm.202013901

Figure Lengend Snippet: The scRNA‐Seq expression of the entire glycolytic pathway was analyzed in primary CD4 + T cells infected in vitro with VSVG‐HIV‐1‐GFP and sorted for viral expression as detailed in Golumbeanu et al . Following latency establishment, cells were left untreated or HIV‐1 expression was reactivated through suberoyl anilide hydroxamic acid (SAHA) or α‐CD3‐CD28 engagement. Clusters 1 (low latency reactivation potential) and 2 (high latency reactivation potential) were identified by principal component analysis as described in Golumbeanu et al . T‐cell subtypes were identified using the SingleR R package and allocated to each cluster irrespective of the in vitro treatment. The expression level of the HUMAN‐GLYCOLYSIS pathway was calculated as the average expression of genes comprising the gene list. n = 1 donor, 131 cells (Cluster 1), 93 cells (Cluster 2).

Article Snippet: Briefly, naive CD4 + T cells were isolated using a RoboSep CD4 + Naïve T‐cell negative selection kit (STEMCELL Technologies Inc., Vancouver, British Columbia, Canada), and 2 × 10 6 cells were resuspended in 10 ml RPMI medium and stimulated with 10 μg/ml concanavalin A (ConA) (EMD Millipore, Billerica, MA, USA) in the presence of subset‐specific cytokines.

Techniques: Expressing, Infection, In Vitro

Journal: EMBO Molecular Medicine

Article Title: Glycolysis downregulation is a hallmark of HIV‐1 latency and sensitizes infected cells to oxidative stress

doi: 10.15252/emmm.202013901

Figure Lengend Snippet: Primary CD4 + T cells were isolated from total blood of healthy donors and activated with α‐CD3‐CD28 beads for 72 h. Cells were then mock‐infected or infected with wild‐type HIV‐1 pNL4‐3 or with Tat‐deficient HIV‐1 pNL4‐3 (Bejarano et al , ). Cells were cultured for 1 week post‐infection, and gene expression was measured by qPCR. A Relative expression of HIV‐1 gag in cells infected with Tat‐deficient HIV‐1 pNL4‐3 as compared to cells infected with wild‐type HIV‐1 pNL4‐3 . B–D Relative expression of the limiting rate enzyme of the pentose phosphate pathway, i.e., G6PD (B), and of genes regulating the thioredoxin and glutathione antioxidant pathways, i.e., TrxR1 (C) and GCLC (D), in HIV‐1 infected as compared to mock‐infected cells. Data information: Data were first normalized using 18S as housekeeping control and then expressed as Log 2 fold mRNA expression in wild type vs Tat‐deficient infection (panel A) or in infected vs mock‐infected cells (panels B‐D), which were calculated using the 2‐ΔΔCT method (Livak & Schmittgen, ). GCLC = glutamate—cysteine ligase; TrxR1= thioredoxin reductase 1. Data are expressed as mean ± SD of two replicates and were analyzed by two‐way ANOVA followed by Sidak’s post‐test. * P < 0.05.

Article Snippet: Briefly, naive CD4 + T cells were isolated using a RoboSep CD4 + Naïve T‐cell negative selection kit (STEMCELL Technologies Inc., Vancouver, British Columbia, Canada), and 2 × 10 6 cells were resuspended in 10 ml RPMI medium and stimulated with 10 μg/ml concanavalin A (ConA) (EMD Millipore, Billerica, MA, USA) in the presence of subset‐specific cytokines.

Techniques: Isolation, Infection, Cell Culture, Gene Expression, Expressing, Control

Journal: EMBO Molecular Medicine

Article Title: Glycolysis downregulation is a hallmark of HIV‐1 latency and sensitizes infected cells to oxidative stress

doi: 10.15252/emmm.202013901

Figure Lengend Snippet: A, B Reactivation from HIV‐1 latency (A) and relative cell viability (B) in primary Th17 cells following 24 h treatment with the Trx inhibitor auranofin (AF; 500 nM), the GSH inhibitor buthionine sulfoximine (BSO; 250 μM), or a combination of the two. GFP‐HIV‐1 expression was determined by FACS. Data are expressed as mean ± SD of three replicates and were analyzed by one‐way ANOVA followed by Tukey's post‐test (A) or two‐way ANOVA followed by Sidak´s post‐test (B). Solid lines represent the means. C Levels of integrated HIV‐1 DNA following treatment for 48 h with AF and/or BSO in CD4 + T cells derived from PLWH under suppressive antiretroviral therapy. Live cells were sorted after treatment, and integrated DNA was measured by Alu‐PCR. The latency reactivating agent SAHA was used as a reference compound (Archin et al , ). Data were analyzed by non‐parametric Friedman´s test followed by Dunn's post‐test. Data information: * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Article Snippet: Briefly, naive CD4 + T cells were isolated using a RoboSep CD4 + Naïve T‐cell negative selection kit (STEMCELL Technologies Inc., Vancouver, British Columbia, Canada), and 2 × 10 6 cells were resuspended in 10 ml RPMI medium and stimulated with 10 μg/ml concanavalin A (ConA) (EMD Millipore, Billerica, MA, USA) in the presence of subset‐specific cytokines.

Techniques: Expressing, Derivative Assay

Journal: bioRxiv

Article Title: Glycolysis downregulation is a hallmark of HIV-1 latency and sensitizes infected cells to oxidative stress

doi: 10.1101/2020.12.30.424810

Figure Lengend Snippet: Panels A-C) primary CD4 + T-cells were activated with α-CD3-CD28 beads and infected with HIV-1 pNL4-3 or mock-infected. Cells were cultured for two weeks post-infection (p.i.) to model different infection stages (days 3-9 p.i., i . e . productive infection; day 14 p.i., i . e . latent infection) and subjected to microarray (A,B; n=2) or RNA-Seq (C; n=3) analysis. Panel A) Gene set enrichment analysis (GSEA) of the expression of the glycolytic pathway (HUMAN-GLYCOLYSIS) in mock-infected or HIV-1 infected cells. B,C) Heatmaps of the relative expression of glycolytic enzymes upon HIV-1 infection. Data are expressed as Log 2 fold change in HIV-1 infected vs mock-infected cells. For microarray data (B) expression values of infected and mock-infected cells at different time points were pooled. For RNA-Seq data (C), expression values in infected cells were normalized using the corresponding time point in mock-infected cells. Adjusted p values (q values) to account for multiple testing were calculated by Significance Analysis of Microarrays [SAM ( Tusher et al , 2001 )] and Deseq2 for RNA-Seq data ( Love et al , 2014 ). Panels D-F) scRNA-Seq of the expression of the entire glycolytic pathway or of glucose phosphate isomerase only ( GPI ) in primary CD4 + T-cells infected in vitro (D,E) or CD4 + T-cells of PLWH (F). In panels D,E, cells were infected with VSVG-HIV-1-GFP and sorted for viral expression as detailed in ( Golumbeanu et al , 2018 ). Following latency establishment, cells were left untreated or HIV-1 expression was reactivated through suberoyl anilide hydroxamic acid (SAHA) or α-CD3-CD28 engagement. Clusters 1 and 2 were identified by principal component analysis as described in ( Golumbeanu et al , 2018 ). In panel F, CD4 + T-cells were isolated from total blood of PLWH under ART as described in ( Cohn et al , 2018 ). Viral expression was reactivated by treatment with phytohemagglutinin (PHA) and cells were sorted using antibodies against Env and Gag. Sorted cells were then subjected to scRNA-Seq analysis. The expression level of the HUMAN-GLYCOLYSIS pathway in Panel D was calculated as the average expression of genes comprising the gene list; expression levels in cluster 1 and 2 were compared using Wilcoxon rank sum test. For panels E-F significance of GPI differential expression level between clusters (E) or between control and Env + Gag + conditions (F) was assessed by Wilcoxon rank sum test encoded in FindMarkers Seurat R function. Panels G,H) Correlation of combined HIV-1 expression and the expression of the entire glycolytic pathway (G) or GPI only (H) in sc-RNA-Seq profiling of untreated HC69 microglial cells. Data were analyzed by Spearman’s correlation coefficients. ** p< 0.01, *** p< 0.001; *** p< 0.0001.

Article Snippet: Briefly, naive CD4 + T cells were isolated using a RoboSep CD4 + Naïve T cell negative selection kit (STEMCELL Technologies Inc., Vancouver, British Columbia, Canada), and 2□X□10 6 cells were resuspended in 10□mL RPMI medium and stimulated with 10□μg/mL concanavalin A (ConA) (EMD Millipore, Billerica, MA, USA) in the presence of subset-specific cytokines.

Techniques: Infection, Cell Culture, Microarray, RNA Sequencing, Expressing, In Vitro, Isolation, Quantitative Proteomics, Control

Journal: bioRxiv

Article Title: Glycolysis downregulation is a hallmark of HIV-1 latency and sensitizes infected cells to oxidative stress

doi: 10.1101/2020.12.30.424810

Figure Lengend Snippet: Panels A,B. Reactivation from HIV-1 latency (A) and relative cell viability (B) in different cell models following treatment with the Trx inhibitor auranofin (AF; 500 nM), the GSH inhibitor buthionine sulfoximine (BSO; 250 μM), or a combination of the two. The characteristics of the different models adopted are detailed in the Material and Methods section. Each data point represents a mean from at least two independent experiments conducted in the different models. Replicates of all experiments for each cell model are shown in Additional Files 8, 9 and 11, except for the data of monocyte-derived macrophages which were retrieved from ( Shytaj et al , 2013 ). Panel C. Levels of integrated HIV-1 DNA following treatment for 48 h with AF and/or BSO in CD4 + T-cells derived from PLWH under suppressive antiretroviral therapy. Live cells were sorted after treatment, and integrated DNA was measured by Alu-PCR. The latency reactivating agent SAHA was used as a reference compound ( Archin et al , 2012 ). Data were analyzed by non-parametric Friedman’s test followed by Dunn’s post-test (A,C) or two-way ANOVA followed by Tukey’s post-test (B). Solid lines represent the means. * p< 0.05, ** p< 0.01, *** p< 0.001.

Article Snippet: Briefly, naive CD4 + T cells were isolated using a RoboSep CD4 + Naïve T cell negative selection kit (STEMCELL Technologies Inc., Vancouver, British Columbia, Canada), and 2□X□10 6 cells were resuspended in 10□mL RPMI medium and stimulated with 10□μg/mL concanavalin A (ConA) (EMD Millipore, Billerica, MA, USA) in the presence of subset-specific cytokines.

Techniques: Derivative Assay