supt1 human t cell lines  (ATCC)

Journal: Nucleic Acids Research

Article Title: Hairpin-induced tRNA-mediated (HITME) recombination in HIV-1

doi: 10.1093/nar/gkl226

Figure Lengend Snippet: Evolution of the replication-impaired HIV-lhNef virus. ( A ) SupT1 cells were transfected with different amounts of HIV-lhNef (culture A, 20 µg; B, 10 µg; C, 5 µg; D, 5 µg; E, 1 µg) and as a control 10 µg of the wild-type HIV-1 molecular clone LAI. To select for replication-competent escape variants, cultures were maintained for 73 days. Virus spread was followed by CA-p24 ELISA of the culture supernatant. Virus-induced syncytia were first observed at day 11 in cultures A and B (20 and 10 µg HIV-lhNef) and day 16 in cultures C and D (both 5 µg). ( B ) Cell samples were taken at different time points for analysis of the proviral DNA by PCR across the hairpin insert with primers tTA1 and CN1 (see ). The amplification products were analysed by 1% agarose gel electrophoresis. The culture is indicated above and the day of harvest below the gel. M is a Smart DNA Ladder (Eurogentec). HIV-1 is the wild-type LAI control. HIV-lhNef could not be amplified with this PCR due to the long hairpin structure in its genome. Indicated is the position of the expected PCR product.

Article Snippet: The human T cell line SupT1 was cultured in RPMI 1640 medium (Invitrogen) supplemented with 10% FCS (Hybond), penicillin (100 U/ml) and streptomycin (100 µg/ml) at 37°C and 5% CO 2 .

Techniques: Transfection, Enzyme-linked Immunosorbent Assay, Amplification, Agarose Gel Electrophoresis

Journal: Nucleic Acids Research

Article Title: Hairpin-induced tRNA-mediated (HITME) recombination in HIV-1

doi: 10.1093/nar/gkl226

Figure Lengend Snippet: Virus production and replication of HIV-lhNef and the escape variants. ( A ) Virus production in HEK293T cells. Cells were transfected with the proviral constructs encoding wild-type HIV-1, HIV-lhNef and the AS escape mutants. Virus production was measured by CA-p24 ELISA in the culture supernatant at three days post-transfection. ( B ) Virus replication in SupT1 and PBMC cells. Cells were transfected with proviral DNA constructs and CA-p24 production was measured in the culture supernatant at several days post-transfection.

Article Snippet: The human T cell line SupT1 was cultured in RPMI 1640 medium (Invitrogen) supplemented with 10% FCS (Hybond), penicillin (100 U/ml) and streptomycin (100 µg/ml) at 37°C and 5% CO 2 .

Techniques: Transfection, Construct, Enzyme-linked Immunosorbent Assay

Journal: Nucleic Acids Research

Article Title: Hairpin-induced tRNA-mediated (HITME) recombination in HIV-1

doi: 10.1093/nar/gkl226

Figure Lengend Snippet: Comparison of the replication capacity of the HIV-lhNef escape viruses and continued evolution. ( A ) SupT1 cells were infected with an equimolar mixture of the five AS escape variants (5 ng CA-p24 of AS44, AS19, AS15, AS11 and AS8) that were produced in HEK293T cells. Virus was passaged at the peak of infection. Cellular DNA was extracted at days 5, 12, 16, 22, 27 and 33 post-infection and proviral DNA around the hairpin insert was PCR amplified with primers tTA1 and CN1 (see ). On the right are indicated the Smart DNA Ladder sizes (bp). ( B ) The PCR products from samples obtained at day 5, 22 and 33 were cloned in the pCR2.1 vector, and 18 or 19 clones were sequenced per sample. The composition of the virus mixture is indicated by the number of clones. The input virus names are in boldface. Newly evolved sequence variants are shown in (C). The free energy Δ G value is a thermodynamic stability score of the perfect duplex structure, e.g. 44 bp for AS44 and 11 bp for AS11. ( C ) Sequence alignment of HIV-lhNef escape viruses and deletion variants asNef sequences are bold caps, tRNA sequences are lower-case italics. Homologous sequences between the tRNA and HIV-lhNef at the recombination junctions are boxed in blue. Repeated sequences at the recombination junctions are underlined.

Article Snippet: The human T cell line SupT1 was cultured in RPMI 1640 medium (Invitrogen) supplemented with 10% FCS (Hybond), penicillin (100 U/ml) and streptomycin (100 µg/ml) at 37°C and 5% CO 2 .

Techniques: Infection, Produced, Amplification, Clone Assay, Plasmid Preparation, 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: (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: Retrovirology

Article Title: Construction of doxycyline-dependent mini-HIV-1 variants for the development of a virotherapy against leukemias

doi: 10.1186/1742-4690-3-64

Figure Lengend Snippet: Replication and cell killing capacity of dox-inducible viruses in SupT1 cells . ( Left ) Virus replication of LAI (△), HIV-rtTA (▲), HIV-rtTAΔ6 A (○), HIV-rtTAΔ6 B (◆) and HIV-rtTA without dox (×) was determined by measuring of the supernatant CA-p24 concentration after infection with virus (20 ng CA-p24) in a 5 mL SupT1 culture. ( Right ) The number of cells in each culture was determined by a 30 sec time limit FACS analysis. The cell killing capacity of the viruses was determined as the ratio of SupT1 cells present in the infected culture versus the uninfected control culture.

Article Snippet: The human T lymphocyte cell line SupT1 [ATCC CRL-1942, [ ]] was cultured in RPMI 1640 (Gibco BRL, Gaithersburg, MD) supplemented with 10% (v/v) FCS, 100 U/mL penicillin, and 100 μg/mL streptomycin at 37°C and in 5% CO 2 .

Techniques: Concentration Assay, Infection

Journal: Retrovirology

Article Title: Construction of doxycyline-dependent mini-HIV-1 variants for the development of a virotherapy against leukemias

doi: 10.1186/1742-4690-3-64

Figure Lengend Snippet: Dox regulated replication of the mini-rtTA virus rtTAΔ6 A . SupT1 cells were infected with rtTAΔ6 A virus (1 ng CA-p24). The culture was split and the cells were cultured with dox (upper panels) or without (lower panels). Virus replication was monitored by CA-p24 Elisa on the culture supernatant. At day 9 post infection, both cultures were washed and each culture split into one culture with dox (left panels) and one without (right panels). Filled triangles indicate cultures without dox and open triangles indicate cultures with 1000 ng/mL dox.

Article Snippet: The human T lymphocyte cell line SupT1 [ATCC CRL-1942, [ ]] was cultured in RPMI 1640 (Gibco BRL, Gaithersburg, MD) supplemented with 10% (v/v) FCS, 100 U/mL penicillin, and 100 μg/mL streptomycin at 37°C and in 5% CO 2 .

Techniques: Infection, Cell Culture, Enzyme-linked Immunosorbent Assay

Journal: Retrovirology

Article Title: Construction of doxycyline-dependent mini-HIV-1 variants for the development of a virotherapy against leukemias

doi: 10.1186/1742-4690-3-64

Figure Lengend Snippet: Selective SupT1 killing in SupT1/PBMC co-cultures by mini-rtTA viruses . Infections were started with virus corresponding to 40 ng CA-p24 or mock infected. The FACS dot plot of the initial PBMC + SupT1 cell mixture is in the lower left corner, and the separate cultures are shown above. The gates for CD4+ PBMC (blue), SupT1 (red) and CD8+ PBMC (green) are indicated. The composition of the PBMC + SupT1 culture was followed over time upon infection with the indicated viruses.

Article Snippet: The human T lymphocyte cell line SupT1 [ATCC CRL-1942, [ ]] was cultured in RPMI 1640 (Gibco BRL, Gaithersburg, MD) supplemented with 10% (v/v) FCS, 100 U/mL penicillin, and 100 μg/mL streptomycin at 37°C and in 5% CO 2 .

Techniques: Infection

Journal: Retrovirology

Article Title: HIV-1 latency in actively dividing human T cell lines

doi: 10.1186/1742-4690-5-37

Figure Lengend Snippet: Overview of SupT1 clones with integrated HIV-rtTA provirus

Article Snippet: The human T lymphocytic cell lines SupT1 (ATCC CRL-1942) [ ], MT2, C8166 and Jurkat (ATCC TIB-152) were cultured in advanced RPMI 1640 medium (Gibco BRL, Gaithersburg, MD) supplemented with 1% (v/v) FCS, 40 U/mL penicillin, and 40 μg/mL streptomycin at 37°C and 5% CO 2 .

Techniques: Clone Assay

Journal: Retrovirology

Article Title: HIV-1 latency in actively dividing human T cell lines

doi: 10.1186/1742-4690-5-37

Figure Lengend Snippet: Northern blot analysis of shRNA production in rtTA-shNef SupT1 cell lines . RNAs from the different HIV-rtTA-shNef SupT1 clones were isolated and separated on an agarose gel. After blotting the membrane was probed with a LNA probe against the shNef. Lane M, marker; lane 1, shNef clone B9; lane 2, clone C10 (a negative control cell line without a provirus); lane 3, shNef clone D2; lane 4, shNef clone D11; lane 5, shNef clone F7; lane 6, shNef clone F8; lane 7 and 8 contain positive controls from a transfection with the F-shNef - and F-shNef + plasmid [31]; lane 9 contains a negative empty-vector control; lane 10 in vitro produced shNef RNA.

Article Snippet: The human T lymphocytic cell lines SupT1 (ATCC CRL-1942) [ ], MT2, C8166 and Jurkat (ATCC TIB-152) were cultured in advanced RPMI 1640 medium (Gibco BRL, Gaithersburg, MD) supplemented with 1% (v/v) FCS, 40 U/mL penicillin, and 40 μg/mL streptomycin at 37°C and 5% CO 2 .

Techniques: Northern Blot, shRNA, Clone Assay, Isolation, Agarose Gel Electrophoresis, Marker, Negative Control, Transfection, Plasmid Preparation, In Vitro, Produced

Journal: Retrovirology

Article Title: HIV-1 latency in actively dividing human T cell lines

doi: 10.1186/1742-4690-5-37

Figure Lengend Snippet: Silencing and reactivation in HIV-rtTA SupT1 cell lines . The HIV-rtTA SupT1 cell lines F7 and B9 were induced with dox or with dox in combination with the indicated activators. DMSO is the solvent for genistein and therefore used as an additional control. Statistical significance was determined with a two-tailed student's T test for each combination versus dox alone (GraphPad Prism). Significant changes ( P -value < 0.05) are indicated with asterisks.

Article Snippet: The human T lymphocytic cell lines SupT1 (ATCC CRL-1942) [ ], MT2, C8166 and Jurkat (ATCC TIB-152) were cultured in advanced RPMI 1640 medium (Gibco BRL, Gaithersburg, MD) supplemented with 1% (v/v) FCS, 40 U/mL penicillin, and 40 μg/mL streptomycin at 37°C and 5% CO 2 .

Techniques: Two Tailed Test

Journal: Retrovirology

Article Title: HIV-1 latency in actively dividing human T cell lines

doi: 10.1186/1742-4690-5-37

Figure Lengend Snippet: Latent HIV-1 infection in SupT1 T cells . TNFα-induced reactivation of silent HIV-1 proviruses in SupT1 cells was analyzed by determining the percentage of CA-p24 producing cells by intracellular FACS analysis. (A) After a single round infection with the LAI isolate, the culture was split and one culture activated for 24 h with TNFα and the other used as a control. (B) The control culture was maintained for one week and split again into a TNFα treated and control culture. Statistical significance was determined with a two-tailed student's T test (GraphPad Prism).

Article Snippet: The human T lymphocytic cell lines SupT1 (ATCC CRL-1942) [ ], MT2, C8166 and Jurkat (ATCC TIB-152) were cultured in advanced RPMI 1640 medium (Gibco BRL, Gaithersburg, MD) supplemented with 1% (v/v) FCS, 40 U/mL penicillin, and 40 μg/mL streptomycin at 37°C and 5% CO 2 .

Techniques: Infection, Two Tailed Test

Journal: Retrovirology

Article Title: HIV-1 latency in actively dividing human T cell lines

doi: 10.1186/1742-4690-5-37

Figure Lengend Snippet: Latent HIV-1 and HIV-rtTA infection in SupT1 T cells . TNFα-induced reactivation of silent HIV-1 proviruses in SupT1 cells was analyzed by determining the percentage of CA-p24 producing cells by intracellular FACS analysis. After a single round infection with HIV-1 (LAI isolate) or the HIV-rtTA-Tat wt , each culture was split and either activated for 24 h with TNFα or not. The fold TNFα activation is the percentage CA-p24 positive cells in the culture with TNFα divided by the percentage of CA-p24 positive cells in the control culture.

Article Snippet: The human T lymphocytic cell lines SupT1 (ATCC CRL-1942) [ ], MT2, C8166 and Jurkat (ATCC TIB-152) were cultured in advanced RPMI 1640 medium (Gibco BRL, Gaithersburg, MD) supplemented with 1% (v/v) FCS, 40 U/mL penicillin, and 40 μg/mL streptomycin at 37°C and 5% CO 2 .

Techniques: Infection, Activation Assay