Journal: Cell

Article Title: Cone-shaped HIV-1 capsids are transported through intact nuclear pores

doi: 10.1016/j.cell.2021.01.025

Figure Lengend Snippet:

Article Snippet: Human T lymphoblast cells SupT1-R5 (stably expressing exogenous CCR5 under puromycin selection; a kind gift from Robert Doms, University of Pennsylvania, USA; certified by Eurofins according to DAkkS ISO 9001:2008) were cultivated at 37°C in a humidified incubator with a 5% CO 2 atmosphere, using RPMI 1640 medium with GlutaMAX (ThermoFisher Scientific) supplemented with 10% fetal bovine serum (FBS; Merck), 50 U/ml of penicillin, 50 μg/ml of streptomycin (ThermoFisher Scientific) and 0.3 μg/ml puromycin (Merck).

Techniques: Virus, Recombinant, Plasmid Preparation, shRNA, Control, Software

Journal: PLoS Pathogens

Article Title: HIV-1 Vpr-Induced Apoptosis Is Cell Cycle Dependent and Requires Bax but Not ANT

doi: 10.1371/journal.ppat.0020127

Figure Lengend Snippet: (A) pHR-VPR-G and pHR-VPR-R are bicistronic lentiviral vectors that encode HIV-1 NL4–3 vpr, an internal ribosome entry site, and either the gene for GFP or that for mRFP, respectively; pHR-VPR(R80A) was derived from pHR-VPR (both the mRFP and the GFP versions) by site-directed mutagenesis; DHIV3 is an envelope-truncated (see gray box) version of HIV-1 NL4–3 ; DHIV3-ΔVPR was derived from DHIV-3 by introducing a frameshift mutation in vpr . (B) SupT1 T lymphocytes were transduced by spin-infection in the presence of 10 μg/ml Polybrene with indicated vectors. Mock-infected cells were subjected to spin-infection in the presence of 10 μg/ml Polybrene without virus. Cells were collected at specified time points post-transduction, stained with propidium iodide, and analyzed for DNA content by flow cytometry to determine cell cycle profiles. The percentage of cells transduced with pHR vectors and DHIV3 viruses ranged between 70% to 80% and between 65% to 70%, respectively, as determined by mRFP or GFP expression (with pHR vectors) or intracellular p24 staining (with DHIV3 vectors). (C) Caspase activation was measured as an indication of apoptosis. SupT1 cells were infected with indicated vectors, harvested, and incubated with FITC-VAD-FMK. The percentage of caspase-active cells at each time point was measured by flow cytometry. (D) Infected SupT1 cells were lysed, and lysates were fractionated into mitochondrial (m) and cytoplasmic (c) fractions and then assayed by Western blot. Western blots were probed with antibodies specific to Smac to measure release from mitochondria and with anti-VDAC antibodies to measure mitochondrial contamination in the cytoplasmic fractions. As a positive control for apoptosis, SupT1 cells were treated with 0.8 μg/ml doxorubicin (dox) for 48 h. (E) Examples of flow cytometric analysis of caspase activation, corresponding to the 72-h time points from (C).

Article Snippet: For experiments in which viral transduction and measurement of cell cycle/apoptosis were the aims, we used the human T-cell line SupT1, which was maintained in RPMI 1640 (Cambrex BioScience), supplemented with 10% FCS and L-glutamine.

Techniques: Derivative Assay, Mutagenesis, Infection, Virus, Transduction, Staining, Flow Cytometry, Expressing, Activation Assay, Incubation, Western Blot, Positive Control

Journal: PLoS Pathogens

Article Title: HIV-1 Vpr-Induced Apoptosis Is Cell Cycle Dependent and Requires Bax but Not ANT

doi: 10.1371/journal.ppat.0020127

Figure Lengend Snippet: HeLa or Supt1 cells were transduced with pHR-VPR-G and at indicated time points, lysed, and analyzed by Western blot with a phospho-specific antibody that recognizes phosphorylation of H3 at serine-10. Nocodazole (250 ng/ml) and doxorubicin (1 μM) were used as positive and negative controls, respectively.

Article Snippet: For experiments in which viral transduction and measurement of cell cycle/apoptosis were the aims, we used the human T-cell line SupT1, which was maintained in RPMI 1640 (Cambrex BioScience), supplemented with 10% FCS and L-glutamine.

Techniques: Transduction, Western Blot, Phospho-proteomics

Journal: PLoS Pathogens

Article Title: HIV-1 Vpr-Induced Apoptosis Is Cell Cycle Dependent and Requires Bax but Not ANT

doi: 10.1371/journal.ppat.0020127

Figure Lengend Snippet: (A) SupT1 cells were infected with pHR-VPR-R or indicated mutants, at an MOI of 0.5. At 48 h postinfection, cells were stained with hypotonic PI to determine the cell cycle profiles. At 72 h postinfection, cells were incubated with FITC-VAD-FMK and analyzed by flow cytometry to determine the percentage of cells with active caspases. (B) Cells from above treatments were lysed at 48 h postinfection, and Western blot was performed to assay for phosphorylation of BRCA1 at Ser1423 by the ATR kinase. To establish the role of ATR in BRCA1 phosphorylation, parallel infections were treated with caffeine (2 mM). (C) Induction of apoptosis by Vpr-GFP and Vpr(R80A)-GFP fusion proteins. HPB-ALL cells were transfected with indicated constructs or mock-transfected and 48 after transfection, phosphatidylserine exposure was analyzed by flow cytometry using phycoerythrin-conjugated annexin V.

Article Snippet: For experiments in which viral transduction and measurement of cell cycle/apoptosis were the aims, we used the human T-cell line SupT1, which was maintained in RPMI 1640 (Cambrex BioScience), supplemented with 10% FCS and L-glutamine.

Techniques: Infection, Staining, Incubation, Flow Cytometry, Western Blot, Phospho-proteomics, Transfection, Construct