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: bioRxiv

Article Title: Direct capsid labeling of infectious HIV-1 by genetic code expansion allows detection of largely complete nuclear capsids and suggests nuclear entry of HIV-1 complexes via common routes

doi: 10.1101/2021.09.14.460218

Figure Lengend Snippet: (a) Activated CD4 + T cells were infected with HIV-1*CA14 SiR (MOI∼0.8) for 24 h before DMSO/PF74 treatment for 1 h, fixation, and methanol extraction. Samples were immunostained against CA (green) and laminA (blue). Images show a single z slice through the cell. Enlargements show the particle marked by the arrowhead. Scale bars: 10 µm (overview) and 1 µm (enlargement). (b) Data analyzed from the experiment outlined in (a). The graph shows the number of CA positive foci per nucleus in cells infected with HIV-1* (n=35 cells, mean=0.85) or HIV-1*CA14 SiR (n= 73 cells, mean=0.51). Pooled data from 6 different blood donors are shown. Grey lines show median and interquartile lines. (c) CA(SiR) intensities of nuclear objects in infected and activated CD4 + T cells at an MOI∼0.8 (n=13; mean=12,485 ± 7,445 a.u.) and an MOI ∼8 (n=7; mean=39,502 ± 18,025 a.u.). MOI was determined in TZM-bl cells. Grey lines show median and interquartile lines. (d-f) Nuclear cone-shaped capsids detected by CLEM-ET. SupT1 cells were treated with 1 µM aphidicolin (APC) for 16 h to prevent cell division, before infection with HIV-1*CA14 SiR virions (2.3 µU RT/cell, corresponds to an MOI∼0.4 determined in TZM-bl cells). At 24 h p.i., cells were cryo-immobilized by high-pressure freezing, freeze substituted, and further processed for CLEM and ET as described in materials and methods. (d) SDCM image of a 250-nm thick resin section of the cell infected with HIV-1*CA14 SiR virions (magenta), post-stained with Hoechst (blue) and decorated with multi-fluorescent fiducials (Fd) for correlation. The arrowhead in the enlargement of the boxed region indicates a CA(SiR) signal within the Hoechst-stained nuclear region. Scale bars: 1 µm (overview) and 200 nm (enlargement). (e) Computational slices through tomographic reconstructions at the correlated region boxed in (d) with views highlighting the presence of clustered capsid-reminiscent structures (black arrowheads) in the nuclear region. Nu, nucleus; NPC, nuclear pore complex; NE, nuclear envelope. Scale bar: 100 nm. (f) Segmented and isosurface rendered structure of the cones detected in (e). Magenta: capsid, yellow: NE, cyan: NPC. See also supplementary movie 1.

Article Snippet: SupT1 cells were distributed in a 96-well plate (2×10 5 cells/well; U-bottom; Greiner Bio-one, 650180) and pre-incubated for 16 h with 1µm aphidicolin (APC; Merck).

Techniques: Infection, Staining

Journal: Scientific Reports

Article Title: Tunable control of CAR T cell activity through tetracycline mediated disruption of protein–protein interaction

doi: 10.1038/s41598-021-01418-9

Figure Lengend Snippet: A split CAR associates through the interaction of TetRB and TIP and dissociates upon minocycline addition. ( a ) Overview of the split CAR approach (TetCAR), incorporating the tetracycline repressor protein B (TetRB) and the peptide TIP. Addition of the small molecule antibiotic minocycline reversibly disrupts TetRB-TIP binding, displaces the endodomain and inhibits CAR activation. ( b ) Schematic of the CAR constructs with eGFP endodomains. CARs contain an anti-human CD19 scFv from FMC63, CD8 stalk regions, CD28 transmembrane domains and eGFP endodomain. TetCARs have a TetRB endodomain with eGFP as a separate protein with or without TIP. ( c ) Representative widefield fluorescent images of HEK293T cells transduced with eGFP-tagged CAR structures, ± 100 nM minocycline. ( d ) Schematic of the CAR constructs with 41BB-CD3ζ endodomains. CARs contain an anti-human CD19 scFv from FMC63, with a CD8 stalk and transmembrane domain and 41BB-CD3ζ endodomain. TetCARs have a TetRB endodomain with 41BB-CD3ζ as a separate protein, with or without TIP. E) Killing of SupT1 cells engineered to express CD19 and GFP (SupT1-CD19-GFP) after 24 h co-culture with CAR-T cells at a 1:1 effector:target ratio. 100 nM of minocycline was added to relevant wells. Data shows mean percentage (± SD) of live cells compared to non-transduced (NT) T-cell control, n = 4 donors from 1 experiment. Statistical analysis was through a two-way ANOVA with Tukey’s multiple comparisons between each group at 0 nM, or with Šidák’s multiple comparisons within each group ± minocycline. P values = FMC63-Tet-BBz 0 nM versus 100 nM (****, < 0.0001). ( f ) IFN-γ or ( g ) IL-2 release after 24 h of co-culture with SupT1-CD19-GFP at 1:1 E:T ratio. Data shows mean ± SD, n = 4 donors from 1 experiment. Statistical analysis was through a two-way ANOVA with Tukey’s multiple comparisons between FMC63-Tet-BBz and TIP-less-Tet-BBz. P values = FMC63-Tet-BBz 0 nM versus 100 nM (**, 0.0013) and FMC63-Tet-BBz 0 nM versus TIP-less-Tet-BBz 0 nM (***, 0.0001).

Article Snippet: SupT1 cells were purchased from the European Collection of Authenticated Cell Cultures.

Techniques: Binding Assay, Activation Assay, Construct, Transduction, Co-Culture Assay, Control

Journal: Scientific Reports

Article Title: Tunable control of CAR T cell activity through tetracycline mediated disruption of protein–protein interaction

doi: 10.1038/s41598-021-01418-9

Figure Lengend Snippet: Optimization of TetCAR surface expression and signaling. ( a ) Schematic overview of TetCAR constructs containing 41BB-ζ or CD28-ζ endodomains. Antigen recognition is provided by the FMC63 scFv or Fab fragment. ( b ) Transduction efficiency as measured by CD34 staining of the RQR8 marker gene. Data shows mean ± SD, n = 5 donors from 2 independent experiments. ( c ) Median fluorescent intensity (left) and representative histograms (right) of CAR expression on surface of RQR8 + cells as measured by staining with soluble, Fc-tagged CD19 protein. Data shows mean ± SD, n = 3 donors from 1 experiment. Unpaired T tests were used for statistical analysis. P values = FMC63-BBz versus Fab-Tet-BBz (***, 0.0003) or Fab-Tet-28z (***, 0.0002), FMC63-Tet-BBz versus Fab-Tet-BBz (ns, 0.089), FMC63-Tet-28z versus Fab-Tet-28z (*, 0.045). ( d ) Killing of SupT1-CD19-GFP after 24 h co-culture with CAR-T cells at a 1:1 effector:target ratio. 100 nM of minocycline was added to relevant wells. Data shows mean percentage (± SD) of live cells compared to non-transduced (NT) control, n = 5 donors from 2 independent experiments. Statistical analysis was through a two-way ANOVA with Šidák’s multiple comparisons within each group ± minocycline. P values for each construct + /- minocycline were: FMC63-Tet-BBz (**, 0.0023), FMC63-Tet-28z (***, 0.0003) and Fab-Tet-28z (*, 0.0279). ( e ) IFN-γ and ( f ) IL-2 release after 24 h of co-culture with SupT1-CD19-GFP at 1:1 E:T ratio. Data shows mean ± SD, n = 5 donors from 2 independent experiments. Statistical analysis was through two-way ANOVAs between the TetCAR groups at 0 nM minocycline (with Tukey’s multiple comparisons) or within these groups ± minocycline (with Šidák’s multiple comparisons). P values were; between FMC63-Tet-BBz and Fab-Tet-BBz (**, 0.0097, IFN-γ) and between FMC63-Tet-28z and Fab-Tet-28z (****, < 0.0001, IFN-γ and **, 0.0017, IL-2). P values between the TetCAR constructs ± minocycline were: FMC63-Tet-BBz (***, 0.0007, IFN-γ), FMC63-Tet-28z (*, 0.0409, IFN-γ), Fab-Tet-BBz (****, < 0.0001, IFN-γ and *, 0.0152, IL-2) and Fab-Tet-28z (****, < 0.0001 both IFN-γ and IL-2).

Article Snippet: SupT1 cells were purchased from the European Collection of Authenticated Cell Cultures.

Techniques: Expressing, Construct, Transduction, Staining, Marker, Co-Culture Assay, Control

Journal: Scientific Reports

Article Title: Tunable control of CAR T cell activity through tetracycline mediated disruption of protein–protein interaction

doi: 10.1038/s41598-021-01418-9

Figure Lengend Snippet: Reconfiguration of endodomain positions enhances TetCAR function. ( a ) Schematic overview of Fab-TetCAR constructs containing membrane-proximal 41BB or CD28 endodomains, with a TIP-CD3ζ or TIP-41BB-CD3ζ domains. ( b ) Transduction efficiency as measured by CD34 staining of the RQR8 marker gene. Data shows mean ± SD, n = 5 donors from 2 independent experiments. ( c ) Median fluorescent intensity of CAR expression on surface of RQR8 + cells as measured by staining with soluble, Fc-tagged CD19 protein. Data shows mean ± SD, n = 5 donors from 2 experiments. Statistical analysis was through one-way ANOVA between the groups, p values were; between FMC63-BBz and each Fab-TetCAR (****, < 0.0001). Differences between Fab-TetCARs alone were analyzed by one-way ANOVA but were not significant. ( d ) Killing of SupT1-CD19-GFP after 24 h co-culture with CAR-T cells at 1:1 E:T ratio. 100 nM of minocycline was added to relevant wells. Data shows mean ± SD, n = 5 donors from 2 independent experiments. Statistical analysis was through a two-way ANOVA comparing each group ± minocycline (with Šidák’s multiple comparisons). P values were; FMC-Tet-BBz (*, 0.0119). ( e ) IFN-γ and ( f ) IL-2 release after 24 h of co-culture with SupT1-CD19 at 1:1 E:T ratio (± 100 nM minocycline). Data shows mean ± SD, n = 5 donors from 2 independent experiments. Statistical analysis was through a 2-way ANOVA comparing each group ± minocycline (with Šidák’s multiple comparisons). P values for IFN-γ = 28-Tet-z (*, 0.0484), 28BB-Fab-Tet-z and 28BB-Fab-Tet-BBz (****, < 0.0001). P values for IL-2 = 28-Tet-z (*, 0.0150), 28BB-Fab-Tet-z (****, < 0.0001), 28-Fab-Tet-BBz (**, 0.0029) and 28BB-Fab-Tet-BBz (***, 0.0007). ( g ) Killing of NALM6 after 48 h co-culture with CAR-T cells at 1:1 E:T ratio. 100 nM of minocycline was added to relevant wells. Data shows mean ± SD, n = 5 donors from 2 independent experiments. Statistical analysis was through a two-way ANOVA comparing each group ± minocycline (with Šidák’s multiple comparisons). P values were; BB-Fab-Tet-z (**, 0.0083), BB-Fab-Tet-BBz (*, 0.0189) and 28-Fab-Tet-z, 28BB-Fab-Tet-z, 28-Fab-Tet-BBz and 28BB-Fab-Tet-BBz (****, < 0.0001). ( h ) IFN-γ and I) IL-2 release after 48 h of co-culture with NALM6 at 1:1 E:T ratio (± 100 nM minocycline). Data shows mean ± SD, n = 5 donors from 2 independent experiments. Statistical analysis was through a 2-way ANOVA comparing each group ± minocycline (with Šidák’s multiple comparisons). P values for IFN-γ = BB-Fab-Tet-z (*, 0.0433), 28-Fab-Tet-z (*, 0.0387), 28BB-Fab-Tet-z (****, < 0.0001) and 28BB-Fab-Tet-BBz (**, 0.0020). P values for IL-2 = 28BB-Fab-Tet-z (*, 0.0450).

Article Snippet: SupT1 cells were purchased from the European Collection of Authenticated Cell Cultures.

Techniques: Construct, Membrane, Transduction, Staining, Marker, Expressing, Co-Culture Assay

Journal: Scientific Reports

Article Title: Tunable control of CAR T cell activity through tetracycline mediated disruption of protein–protein interaction

doi: 10.1038/s41598-021-01418-9

Figure Lengend Snippet: TetCAR activity can be fine-tuned in vitro with a dose-dependent response to minocycline. ( a ) Killing of SupT1-CD19-GFP after 24 h of co-culture with CAR-T cells at 1:4 E:T ratio. A range of minocycline doses from 0.02-1600 nM were added to relevant wells. Data shows mean % of live targets relative to an inert TetCAR control, ± SD. n = 4 donors from 1 experiment. ( b ) IFN-γ and ( c ) IL-2 release after 24 h of co-culture with SupT1-CD19-GFP at various minocycline doses. Data shows mean ± SD, n = 4 donors from 1 experiment. ( d ) IL-2 secretion from FMC63-BBz or 28BB-Fab-Tet-z CARs 1–5 h after co-culture with SupT1-CD19-GFP at a 2:1 E:T ratio. 100 nM minocycline was added to separate wells every hour. Data shows the mean (± SD) secretion of IL-2 at each time-point in groups that received minocycline at the beginning of the experiment, or every hour afterwards. Color coded bars indicate the number of hours that the co-cultures were exposed to minocycline for. n = 4 donors from 2 independent experiments. ( e ) Cytotoxicity or ( f ) IL-2 secretion by 28BB-Fab-Tet-z CARs after coculture with SupT1-CD19 at a 1:1 E:T ratio. Inhibition by minocycline was removed by washing cells with complete media at 48, 24 and 2 h before addition of SupT1-CD19 targets. Wash steps are indicated by “[W]”. Data shows mean (± SD) % of live targets relative to NT T cells ( e ) or mean (± SD) IL-2 secretion ( f ) after 24 h. n = 3 donors from 1 experiment. Statistical analysis was through a one-way ANOVA with multiple comparisons between the 28BB-Fab-Tet-z CAR under different conditions. P values for cytotoxicity ( e ) were: 48 h wash versus no wash (*, 0.0118), 24 h wash versus no wash (**, 0.0050) and no drug versus no wash (**, 0.0044). P values for IL-2 secretion ( f ) were: 48 h wash versus no wash (**, 0.0015), no drug versus no wash (**, 0.0044), no drug versus 2 h wash (*, 0.0103) and 48 h wash versus 2 h wash (**, 0.0033).

Article Snippet: SupT1 cells were purchased from the European Collection of Authenticated Cell Cultures.

Techniques: Activity Assay, In Vitro, Co-Culture Assay, Control, Inhibition

Journal: Scientific Reports

Article Title: Tunable control of CAR T cell activity through tetracycline mediated disruption of protein–protein interaction

doi: 10.1038/s41598-021-01418-9

Figure Lengend Snippet: Effector function of CD28-containing TetCAR matches 41BBζ control CAR. ( a ) Killing of SupT1-CD19-GFP after 24 h or NALM6 after 48 h of co-culture with CAR-T cells at 1:1–1:32 E:T ratio. Data shows mean ± SD, n = 4 donors from 2 independent experiments. ( b ) SupT1-CD19, NALM6, Raji or Raji-CD19KO targets were incubated with mitomycin C, then co-cultured with CAR-T cells at 1:2 E:T ratio for 7 days. To relevant wells, 400 nM of minocycline was added on day 0. Graphs show mean (± SD) number of RQR8 + T cells (filled bars) or total CD3 + T cells (white bars) for each target. n = 4 donors (SupT1-CD19, Raji and Raji-CD19KO) or n = 3 (NALM6) from 2 independent experiments. Statistical analysis was through a two-way ANOVA comparing mean RQR8 number in each group ± minocycline (with Šidák’s multiple comparisons). P values for SupT1-CD19 were 28-Fab-Tet-z (**, 0.0028) and 28BB-Fab-Tet-z (**, 0.0050). P values for Raji were 28-Fab-Tet-z (*, 0.0320) and 28BB-Fab-Tet-z (*, 0.0304). ( c ) Mean fluorescent intensity of Tim3 and Lag3 after 7 days coculture with SupT1-CD19 targets, ± 400 nM minocycline. Data shows geometric mean (± SD) in CD3 + T cells. n = 4 donors, from 2 independent experiments. Statistical analysis was through a two-way ANOVA comparing each group ± minocycline (with Šidák’s multiple comparisons). P values for Lag3 expression were 28-Fab-Tet-z (**, 0.0078) and 28BB-Fab-Tet-z (*, 0.0207). ( d ) Percentage of naïve (CD62L + , CD45RA + ), Tcm (central memory; CD62L + , CD45RA - ), Tem (effector memory; CD62L - , CD45RA - ) or Temra (terminally differentiated effector memory; CD62L - , CD45RA + ) memory T cell populations after 7 days coculture with SupT1-CD19 targets, ± 400 nM minocycline. Data shows mean (± SD) in CD3 + T cells. n = 4 donors, from 2 independent experiments.

Article Snippet: SupT1 cells were purchased from the European Collection of Authenticated Cell Cultures.

Techniques: Control, Co-Culture Assay, Incubation, Cell Culture, Expressing

Journal: PLoS Biology

Article Title: The endoplasmic reticulum proteostasis network profoundly shapes the protein sequence space accessible to HIV envelope

doi: 10.1371/journal.pbio.3001569

Figure Lengend Snippet: (A) Chemical genetic strategy to orthogonally regulate XBP1s and ATF6 in SupT1 DAX cells. (B–D) RNA sequencing (RNA-Seq) analysis of the transcriptomic consequences of (B) XBP1s, (C) ATF6, and (D) XBP1s/ATF6 induction. Transcripts that were differentially expressed under each condition based on a >1.5-fold change in expression level (for dox-, TMP-, or dox- and TMP-treated versus vehicle-treated cells) and a non-adjusted p -value < 10 −10 are separated by dashed lines and plotted in red, with select transcripts labeled. The lowest nonzero p -value recorded was 10 −291 ; therefore, p -values equal to 0 were replaced with p -value = 1.00 × 10 −300 for plotting purposes. Transcripts for which p -values could not be calculated owing to extremely low expression or noisy count distributions were excluded from plotting. (E–G) Comparison of transcript fold change upon (E) +XBP1s versus +ATF6, (F) +ATF6 versus +XBP1s/+ATF6, and (G) +XBP1s versus +XBP1s/+ATF6 remodeling of the endoplasmic reticulum proteostasis network. Only transcripts with false-discovery-rate-adjusted p -value < 0.05 and fold increase > 1 in both of the indicated conditions are plotted. Dashed lines indicate a 1.5-fold filter to assign genes as selectively induced by the proteostasis condition on the x -axis (red), y -axis (blue), or lacking selectivity (purple). Transcripts with fold increase < 1.2 in either proteostasis environment are colored in grey to indicate low differential expression. The complete RNA-Seq differential expression analysis is provided in . dox, doxycycline; TMP, trimethoprim.

Article Snippet: With stably engineered SupT1 DAX cells in hand, we anticipated that we could create 4 distinct ER proteostasis environments (basal, XBP1s-induced, ATF6-induced, and XBP1s/ATF6 co-induced) to assess potential consequences for Env mutational tolerance.

Techniques: RNA Sequencing, Expressing, Labeling, Comparison, Quantitative Proteomics

Journal: PLoS Biology

Article Title: The endoplasmic reticulum proteostasis network profoundly shapes the protein sequence space accessible to HIV envelope

doi: 10.1371/journal.pbio.3001569

Figure Lengend Snippet: (A) Scheme for deep mutational scanning of Env in 4 distinct ER proteostasis environments (basal, +XBP1s, +ATF6, and +XBP1s/+ATF6). SupT1 DAX cells were pretreated with DMSO (basal), dox (+XBP1s), TMP (+ATF6), or both dox and TMP (+XBP1s/+ATF6) 18 h prior to infection with biological triplicate Env viral libraries. 4 d post-infection, cells were harvested, and non-integrated viral DNA was sequenced to quantify the diffsel of Env variants. (B) Diffsel for each amino acid variant can be visualized in a sequence logo plot. The black horizontal lines at the center represent the diffsel for the wild-type amino acid at that site, and the height of the amino acid letter abbreviations is proportional to the diffsel of that variant in the remodeled ER proteostasis environment relative to the basal environment. Variants that are relatively enriched in the indicated ER proteostasis environment (positive diffsel) are located above the black horizontal line. Variants that are relatively depleted in the indicated ER proteostasis environment (negative diffsel) are located below the black horizontal line. (C) Net site diffsel for all Env sites in 3 perturbed ER proteostasis environments, averaged over biological triplicates. The black horizontal lines on the violin plots indicate the median (solid line) and the first and the third quartiles (dashed lines) of the distribution. The significance of deviation from null (net site diffsel = 0, no selection) was tested using a 1-sample t test, with 2-tailed p -values shown. The mean of the distribution and the number of sites with net site diffsel >0 or <0 are listed below the distribution. (D and E) Correlation for net site diffsel values for (D) +XBP1s/+ATF6 versus +XBP1s and (E) +XBP1s/+ATF6 versus +ATF6, normalized to the basal proteostasis environment. Pearson correlation coefficients ( r ) and corresponding p -values are shown. Select sites with highly positive or highly negative net site diffsel values in both proteostasis environments are marked in red and labeled with site numbers. (F) Diffsel for individual Env variants in 3 perturbed ER proteostasis environments, averaged over biological triplicates. The black horizontal lines on the violin plots indicate the median (solid line) and the first and the third quartiles (dashed lines) of the distribution. The significance of deviation from null (diffsel = 0, no selection) was tested using a 1-sample t test, with 2-tailed p -values shown. The mean of the distribution and the number of sites with diffsel >0 and <0 are listed below the distribution. Diffsel values (C–F) are provided at https://github.com/yoon-jimin/2021_HIV_Env_DMS . diffsel, differential selection; dox, doxycycline; ER, endoplasmic reticulum; TMP, trimethoprim; WT, wild-type.

Article Snippet: With stably engineered SupT1 DAX cells in hand, we anticipated that we could create 4 distinct ER proteostasis environments (basal, XBP1s-induced, ATF6-induced, and XBP1s/ATF6 co-induced) to assess potential consequences for Env mutational tolerance.

Techniques: Infection, Variant Assay, Sequencing, Selection, Labeling

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