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Blood, Vol. 113, Issue 1, 58-65, January 1, 2009
Induction of HIV-1 latency and reactivation in primary memory CD4+ T cells
Blood Bosque and Planelles
113: 58
Supplemental materials for: Bosque and Planelles
Concentration of each inhibitor or activator
The concentration of each inhibitor or activator was as follows: 5 µM H-89, 50 µM PD98059, 250 nM Wortmannin, 15 nM bisindolylmaleimide II (BIM), 5 µM Rottlerin, 10 µM PP2, 50 µg/ml I kappa B Kinase Inhibitor Peptide, Cell-Permeable (IKK inh.) and 50 µM Forskolin (Calbiochem, San Diego, CA); 50 µM SB202190 and 125 ng/ml Leflunomide (Alexis Biochemicals, San Diego, CA); 10 nM Gö6976 and 1 µM Prostratin (LC Laboratories, Woburn, MA); 25 ng/ml TNF-α (Prepotech Inc.); 10 ng/ml phorbol 12-myristate 13-acetate, 1 µM Ionomycin, 5 µg/ml L-PHA and 1 mM Valproic Acid (Sigma, Saint Louis, MO); 500 ng/ml Cyclosporin A (Fluka/Sigma); 25 µM SP600125 (A.G. Scientific Int., San Diego, CA ); and 20 µM Integrase Inhibitor (118-D-24). Direct mutagenesis of HIV-1 LTR
List of primers used to generate the different mutants. Nucleotides mutated are indicated in bold. kB/NFAT-1
Forward 5′-tgacatcgagcttgctacaactcactttccgctggggac-3′
Reverse 5′-gtccccagcggaaagtgagttgtagcaagctcgatgtc-3′
kB/NFAT-2
Forward 5′-aagggactttccgctgctcactttccagggaggcg-3′
Reverse 5′-cgcctccctggaaagtgagcggaaagtccctt-3′
AP-2
Forward 5′-cttgctacaagggactttccatatgggactttccagggaggcgt-3′
Reverse 5′-cgcctccctggaaagtcccatatggaaagtcccttgtagcaag-3′
Sp-1
Forward 5′-ggggactttccagggattcgtggcctgttcgggactggttagtggcgag-3′
Reverse 5′-ctcgccactaaccagtcccgaacaggccacgaatccctggaaagtcccc-3′
USF
Forward 5′-gtttgacagccgcctagcatttcatgaattcgcccgagagctgc-3′
Reverse 5′-gcagctctcgggcgaattcatgaaatgctaggcggctgtcaaac-3′
TCF-1α
Forward 5′-gctgcatccggagtacgaattcaactgctgacatcgagc-3′
Reverse 5′-gctcgatgtcagcagttgaattcgtactccggatgcagc-3′
NFIL6-I
Forward 5′-ttgacagccgcctagcatttaatcacgtggcc-3′
Reverse 5′-ggccacgtgattaaatgctaggcggctgtcaa-3′
NFIL6-II
Forward 5′-cttcaagaactgctgacatcgagagctgtacaagggactttccgctgggga-3′
Reverse 5′-tccccagcggaaagtcccttgtacagctctcgatgtcagcagttcttgaag-3′
Files in this Data Supplement:
- Figure S1. Phenotypic analysis of T cells (JPG, 118 KB)
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Naïve cells were primed in NP, Th1- or Th2-polarizing conditions and were subject to an extensively phenotypic analysis. Data are representative of analysis performed with 5 different donors. (A) Activation of naïve T cells led to expression of the activation markers, CD69, CD25 and HLA-DR: CD69 (early activation marker) and CD25 (medium-time activation marker) were analyzed 3 days after activation. HLA-DR (late activation marker) was analyzed 7 days after activation. (B) HIV-1 receptor, CD4, and co-receptors, CXCR4 and CCR5, were analyzed at the time of infection (7 days after activation) and compared with naïve cells. (C) CD45RA and CD45RO were analyzed at the time of infection and compared with naïve cells. The subset analyzed by each marker is indicated between parentheses. (D) Cells were analyzed for the expression of CCR7 and CD27, surface markers expressed in naïve and central memory T cells at day 0, 7, 14 and 21-post activation. (E) The phenotype of Th1 and Th2 cells was confirmed via intracellular staining for IFN-γ and IL-4, respectively 7 days after activation. On day 7, cells were restimulated with PMA plus Ionomycin for 1 h plus an additional 3h in the presence of brefeldin A for intracellular cytokine detection. Also, cells were analyzed for the expression of CrTH2, surface marker expressed in Th2.
- Figure S2. p24Gag intracellular staining correlates with GFP expression (JPG, 60.1 KB)
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Cells were primed in NP conditions and 7 days after activation cells were non infected (Mock), infected with DHIV (DHIV Infected) or infected with a DHIV in which nef has been replaced by GFP (DHIV-GFP Infected). 3 days after infection cells were assessed for intracellular p24Gag and GFP expression by flow cytometry. 7 days after infection cells were cultured without stimulation (untreated) or co-stimulated with antibodies to CD3 and CD28 for 3 days (CD3/CD28) and assessed for intracellular p24Gag and GFP expression by flow cytometry. The percentage of cells is indicated in each panel for this representative experiment.
- Figure S3. T-cell signaling (JPG, 99.1 KB)
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Diagram to describe the main T-signaling pathways analyzed in this work. The inhibitors and agonist used in this work are represented with red letters or green letters, respectively.
- Figure S4. Panel of LTR mutants (JPG, 92.4 KB)
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Scheme representing the different mutants generated within the HIV-1 LTR. En each one, the nucleotides mutated are represented with bold letters.
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