human cd4 t cell line supt1  (ATCC)

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: PLoS ONE

Article Title: HIV-1 Vpr Triggers Mitochondrial Destruction by Impairing Mfn2-Mediated ER-Mitochondria Interaction

doi: 10.1371/journal.pone.0033657

Figure Lengend Snippet: A , HEK293 cells were transfected with GFP vector the plasmid encoding Vpr-GFP or Vpr 52–96- GFP, and harvested at different time (hours) post-transfection for PI staining. The percentage of dead cells among GFP-expressing cells was determined by flow cytometry. *** ( p <0.001) indicates significantly different from the GFP vector control. B , HEK293 cells were infected with lenti-vector (control) or Lenti-Vpr and harvested at different time (hours) post-infection for PI staining. The percentage of dead cells was determined by flow cytometry. *** ( p <0.001) indicates significantly different from the control. C, HEK293 and SupT1 cells were grown in 10% FBS or starved for 24 hours, and infected with Vpr-expressing lentivirus for 48 and 72 hours. The expression of Mfn2 was decreased in serum-starved HEK293 or SupT1 cells. The quantitative expression of Mfn2 was measured by Image J and normalized with the expression of β-actin. C indicates Vpr negative lentiviral control. D, MMP loss was determined after Vpr-expressing lentivirus infection. Vpr significantly impaired MMP in serum-starved HEK293 and SupT1 cells. E, Vpr expression led to cell death in serum-starved HEK293 and SupT1 cells. For panels D and E , results are the means ± S.D. of three independent experiments. * ( p <0.05), ** ( p <0.01) and ** ( p <0.001) indicate significantly higher than the Vpr negative lentiviral control (Con.). † (p<0.05) and †† ( p <0.01) indicate significantly different between 10% FBS and serum starvation. F, Human primary CD4 + T cells were isolated from peripheral blood mononuclear cell (PBMC) and infected with Vpr-expressing lentivirus for 72 hours. The expression of Mfn2 was decreased and the expression of nuclear DRP1 was increased in human primary CD4 + cells. The relative expression levels of Mfn2 and DRP1 were measured by Image J and normalized with the expression of β-actin. G, MMP loss was determined after Vpr-expressing lentivirus infection. Vpr led to a significant MMP loss in human primary CD4 + T cells. H, Vpr expression led to cell death in human primary CD4 + T cells. For panels G and H , results are the means ± S.D. of three independent experiments. ** ( p <0.01) and *** ( p <0.001) indicate significantly higher than control human primary CD4 + T cells. Band intensities were calculated using Image J. Relative intensities are shown at the bottom of each panel.

Article Snippet: Human CD4 + T lymphoblast cell line SupT1 was obtained from ATCC and grown in RPMI1640 medium supplemented with 10% FBS, 4 mM glutamine, 100 U/ml penicillin and 100 µg/ml streptomycin.

Techniques: Transfection, Plasmid Preparation, Staining, Expressing, Flow Cytometry, Infection, Isolation

Journal: PLoS ONE

Article Title: HIV-1 Vpr Triggers Mitochondrial Destruction by Impairing Mfn2-Mediated ER-Mitochondria Interaction

doi: 10.1371/journal.pone.0033657

Figure Lengend Snippet: After transient transfection of Mfn2, or DRP1 expression plasmid for 24 hours, cells were infected with Vpr-expressing lentivirus for 72 hours. A, In contrast to Lenti-Vpr negative (-) HEK293 cells (medium control and vector control), the 89 kDa cleavage fragment of PARP appeared exclusively in Lenti-Vpr positive (+) HEK293 cells. PARP cleavage was reduced in Mfn2-, DRP1- and Mfn2/DRP1-overexpressed Lenti-Vpr (+) HEK293 cells. B, PARP cleavage occurred only in Lenti-Vpr (+) SupT1 cells. The cleavage of PARP was reduced in Mfn2-, DRP1- and Mfn2/DRP1-expressed Lenti-Vpr (+) SupT1 cells. C, Overexpression of Mfn2, DRP1, and Mfn2/DRP1 decreased the percentage of MMP loss in Lenti-Vpr (+) HEK293 and SupT1 cells. D, Overexpression of Mfn2, DRP1, and Mfn2/DRP1 reduced Vpr-induced apoptosis. are the means ± S.D. of three independent experiments. For panels C and D , † (p<0.05) and †† ( p <0.01) indicate significantly lower than Lenti-Vpr (+) cells pretreated with mock (medium control) or vector transfection.

Article Snippet: Human CD4 + T lymphoblast cell line SupT1 was obtained from ATCC and grown in RPMI1640 medium supplemented with 10% FBS, 4 mM glutamine, 100 U/ml penicillin and 100 µg/ml streptomycin.

Techniques: Transfection, Expressing, Plasmid Preparation, Infection, Over Expression

Journal: Virology Journal

Article Title: CHD1 and CHD2 are positive regulators of HIV-1 gene expression

doi: 10.1186/1743-422X-11-180

Figure Lengend Snippet: CHD2 and CHD1 regulate retroviral infection in human cells. (A) siRNAs targeting CHD1 and CHD2 were transfected into HEK293T cells. A non-targeting siRNA (Neg) and a siRNA targeting the virus-encoded luciferase (GL3) were used as controls. (Left panel) Immunoblot analysis of CHD1, CHD2 and tubulin protein levels at 48 hours post-transfection. (Right panel) Protein levels were quantified using the Licor Odyssey infrared imaging system and the ratios of CHD1 or CHD2 protein:tubulin were determined and compared to the non-targeting control (defined as 100% Control). The data shown is the average mean of three independent experiments. (B) HEK293T cells transfected with the siRNAs were challenged with the MLV vector or (C) the HIV-1 vector. The ratios of luciferase activity:cell viability were determined and compared to the non-targeting control (defined as 100% infection). (D) siRNAs targeting both CHD1 and CHD2, or a control siRNA (Neg) were transfected into SupT1 cells. (Right panel) The cells were challenged with the HIV-1 vector as described above. (Left panel) CHD1 and CHD2 mRNA levels were measured by qRT-PCR. The ratios of CHD1 or CHD2 mRNA:GAPDH mRNA were determined and compared to the non-targeting control (defined as 100% Control). (Right panel) The ratio of virus-encoded luciferase activity:viable cell number was determined and compared to the non-targeting control (defined as 100% infection). (E) Immunoblot analysis of CHD1-V5, CHD2-Myc/His and tubulin protein levels at 48 hours post-transfection in transfected HEK293T cells. (F) The cDNA-expressing cells were challenged with the HIV-1 vector at 48 hours post-transfection. The ratio of virus-encoded luciferase activity:viable cell number was determined and compared to the non-targeting control (defined as 100% infection). The data shown is the average mean of three independent experiments each performed with triplicate samples. Error bars indicate the standard deviation. The data was analyzed using an unpaired T-test, ***P value <0.0001.

Article Snippet: The human CD4+ T-cell line SupT1 was cultured in RPMI (Invitrogen) supplemented with 10% FBS (Hyclone).

Techniques: Infection, Transfection, Luciferase, Western Blot, Imaging, Plasmid Preparation, Activity Assay, Quantitative RT-PCR, Expressing, Standard Deviation