jurkat cells  (ATCC)

Journal: bioRxiv

Article Title: Atlas of HIV cis-regulatory elements reveals extensive transcriptional variation across clades, isolates, and within individuals

doi: 10.64898/2026.04.03.716403

Figure Lengend Snippet: (A) Schematic of MPRAs tiling through HIV genomes, saturation mutagenesis, and evaluation of different isolates. (B) Saturation mutagenesis MPRA experiments in unstimulated Jurkat cells for four tiles in the HIV-1 LTR from the clade B REJO strain that show transcriptional activity. Region coordinates are provided using standardized HXB2 genomic coordinates. TF motifs that contribute to activity are outlined. Inset shows MPRA activity tiling through the HIV-1 LTR. Blue indicates activity of tiles in the sense, and red in the antisense (opposite orientation) strand. (C) CASCADE-derived motifs for different cofactors at various regions of the HIV-1 LTR. (D) Regional distribution of HIV-1 isolates tested by MPRAs. (E) Violin plots showing the distribution of activity in unstimulated Jurkat cells across 5,569 isolates for different regions of the HIV-1 LTR across clades. The thick black line indicates the median, and the dotted lines indicate the first and third quartiles. The dots heatmaps below indicate the fold activation by stimulation of Jurkat cells with αCD3+PMA, TNFα, or IFNγ. The color indicates the median fold activation across isolates, whereas the size of the dots reflects the fold activity differences between the 5th and 95th percentiles. (F) Pearson correlation between the activity of different regions of the HIV-1 LTR across isolates. (G) Schematic of chimeric proviral constructs and reactivation experimental approach. (H-I) Fold change in GFP+ cells relative to DMSO control (H) or geometric mean fluorescence intensity of GFP+ cells (I) in Jurkat cells infected with chimeric proviruses carrying the U3 region of the indicated isolates from clades A, B, and C, and stimulated with TNFα for 2 days. (J) Comparison between fold activation of MPRA data from tile HXB2:250-431 in Jurkat cells activated with TNFα, and provirus reactivation by TNFα measured as fold change in GFP+ cells relative to DMSO control or geometric mean fluorescence intensity of GFP+ cells. Pearson correlation coefficients and one-tailed p-values are indicated for each comparison to MPRA data. (K) Dot heatmaps of recombinant HIV-1 viruses derived from clades B and C. Median baseline activity in tile HXB2:250-431 is shown in shades of green, whereas the median fold activation by αCD3+PMA, TNFα, or IFNγ is shown blue-white-red gradient. The size for the dots reflects the fold activity differences between the 5th and 95th percentiles.

Article Snippet: Jurkat cells (ATCC-TIB-15) were cultured in RPMI media (Fisher Scientific, Catalogue # A1049101) with 10% Fetal Bovine Serum (R&D Systems, Catalog # S12450H) and 1% Antibiotic-antimicotic (Thermofisher Scientific, Catalogue #15240062) up to a density of 1 million cells per mL prior to transfection.

Techniques: Mutagenesis, Activity Assay, Derivative Assay, Activation Assay, Construct, Control, Fluorescence, Infection, Comparison, One-tailed Test, Recombinant

Journal: bioRxiv

Article Title: Atlas of HIV cis-regulatory elements reveals extensive transcriptional variation across clades, isolates, and within individuals

doi: 10.64898/2026.04.03.716403

Figure Lengend Snippet: (A) Distribution of the number of TF binding sites across HIV-1 isolates in tiles HXB2:250-431 and HXB2:581-780. Each pie chart shows the proportion of isolates with different number of binding sites for the indicated TFs. (B-C) Violin plots of baseline activity or fold activation by αCD3+PMA or TNFα for tile HXB2:250-431 across HIV-1 isolates based on the number of NF-κB (B) or SP/KLF sites (C). The thick black line indicates the median, and the dotted lines indicate the first and third quartiles. (D, H) Activity distribution in Jurkat cells across HIV-1 isolates with different TF configurations in tiles HXB2:250-431 (D) and HXB2:581-780 (H). The left boxes represent the TF configurations based on aligned TF positions. The distribution of isolates from different clades across TF configurations is shown as pie chats. Violin plots indicate the distributions of activity in unstimulated Jurkat cells (baseline), and cells stimulated with αCD3+PMA, TNFα, or IFNγ. In the case tile HXB2:250-431 only configurations with at least 10 isolates are shown. (E) Alphafold3 model of the HIV-1 REJO LTR including three SP1 and two sets of NF-κB (p65 and p50) proteins. (F-G) Violin plots of baseline activity or fold activation by IFNγ for tile HXB2:581-780 across HIV-1 isolates based on the number of IRF (F) or SP/KLF sites (G). The thick black line indicates the median, and the dotted lines indicate the first and third quartiles. (I) Violin plots of F-statistic showing the variability in activity for each isolate for tile HXB2:581-780 across four donors in CD4+ T cells. The thick black line indicates the median, and the dotted lines indicate the first and third quartiles. (J) Activity distribution and donor variability (F-statistic) in CD4+ T cells in Jurkat cells across HIV-1 isolates with different TF configurations in tile HXB2:581-780. The left boxes represent the TF configurations based on aligned TF positions. The distribution of isolates from different clades across TF configurations is shown as pie charts. (K) Distribution of donor variability (F-statistic) in tile HXB2:581-780 for isolates that contain or lack an SP/KLF site across four CD4+T cell donors or five replicates of Jurkat cells. Statistical significance determined by two-tailed Brunner-Munzel test.

Article Snippet: Jurkat cells (ATCC-TIB-15) were cultured in RPMI media (Fisher Scientific, Catalogue # A1049101) with 10% Fetal Bovine Serum (R&D Systems, Catalog # S12450H) and 1% Antibiotic-antimicotic (Thermofisher Scientific, Catalogue #15240062) up to a density of 1 million cells per mL prior to transfection.

Techniques: Binding Assay, Activity Assay, Activation Assay, Two Tailed Test

Journal: bioRxiv

Article Title: Atlas of HIV cis-regulatory elements reveals extensive transcriptional variation across clades, isolates, and within individuals

doi: 10.64898/2026.04.03.716403

Figure Lengend Snippet: (A) Schematic of CREST and LARM models. CREST predicts baseline activity in Jurkat cells from DNA sequence. LARM predicts fold-activation by αCD3+PMA or TNFα for sequences corresponding to tile HXB2:250-431. (B) Pearson correlation between baseline activity in Jurkat cells measured by MPRAs and predicted using CREST for the validation and test sets. (C) Pearson correlation between TNFα-induced fold activation of tile HXB2:250-431 isolates in Jurkat cells measured by MPRAs and the corresponding values predicted by LARM for the validation and test sets. (D-E) Baseline activity predicted by CREST in tile HXB2:250-431 for isolates heterosexual (D) or mother-to-infant (E) transmission pairs. , The activity of the predicted founder sequence is indicated for infants. (F) Baseline activity predicted by CREST in tile HXB2:250-431 for isolates from 7 PWH from samples obtained at different days post infection. Error bars indicate the standard deviation, dots indicate the average value. (G) Baseline activity predicted by CREST for tile HXB2:250-431 of two PWH. Isolates are shown within their respective evolutionary trees. Node color represents the activity and node size indicates the average day post infection for the corresponding haplotype. Square nodes correspond to predicted ancestral sequences. (H) Baseline activity predicted by CREST for tile HXB2:250-431 for samples from Los Alamos database plotted versus the date of sample collection. Isolates are colored by clade. (I) Fold activation in Jurkat cells stimulated with TNFα predicted by LARM in tile HXB2:250-431 for isolates from 7 PWH from samples obtained at different days post infection. Error bars indicate the standard deviation, dots indicate the average value.

Article Snippet: Jurkat cells (ATCC-TIB-15) were cultured in RPMI media (Fisher Scientific, Catalogue # A1049101) with 10% Fetal Bovine Serum (R&D Systems, Catalog # S12450H) and 1% Antibiotic-antimicotic (Thermofisher Scientific, Catalogue #15240062) up to a density of 1 million cells per mL prior to transfection.

Techniques: Activity Assay, Sequencing, Activation Assay, Biomarker Discovery, Transmission Assay, Infection, Standard Deviation

Journal: bioRxiv

Article Title: Atlas of HIV cis-regulatory elements reveals extensive transcriptional variation across clades, isolates, and within individuals

doi: 10.64898/2026.04.03.716403

Figure Lengend Snippet: (A) MPRA activity map across the genome of HIV-1 clade B REJO strain in unstimulated Jurkat cells. The genome organization is shown below. (B) Saturation mutagenesis MPRA experiments in unstimulated Jurkat cells for HIV-1 intragenic CREs at HXB2:1330-1530 and HXB2:7784-7984. TF motifs that contribute to activity are outlined. (C-D) Violin plots showing the distribution of activity across isolates for intragenic CREs from HIV-1 across clades: HXB2:1330-1530 (C) and HXB2:7784-7984 (D). The thick black line indicates the median, and the dotted lines indicate the first and third quartiles. (E) Entropy decomposition across the ETS–bZIP region in HIV-1 Gag aligned to HXB2 coordinates (codon start positions). Codon entropy (H(codon)), amino-acid entropy (H(a.a.)), and conditional synonymous entropy (H(codon|a.a.)) are shown; the motifs are outlined. (F) Violin plots showing the distribution of Δ median entropy (ROI − rest of window) from circular-shift permutation tests for H(codon), H(a.a.), and H(codon|a.a.). P values are indicated; n.s. denotes not significant. (G) Los Alamos National Laboratory (LANL) genome alignments of HIV-1 strains using the HXB2 genome as a reference. Transcriptional activity predicted using CREST is shown in shades of green. (H) Scatter plot showing the activity predicted using CREST for the two CREs in HIV-1 env . Dots are colored by clade. The top diagram shows the location of the CREs within the env protein coding sequence. C1-5 = conserved regions, V1-5 = variable regions, FP = fusion peptide. (I) TF motif profiles determined using CREST-based saturation mutagenesis across HIV-1 isolates from LANL. Regions were aligned to the HXB2 genome. (J-K) Baseline activity in Jurkat cells for two PWH predicted using CREST corresponding to HXB2 regions 7432-7632 (J) and 7712-7912 (K). Isolates are shown within their respective evolutionary trees. The color of the nodes represents activity levels. The size of the nodes indicate the day post infection of the corresponding sample. Square nodes represent predicted ancestral sequences.

Article Snippet: Jurkat cells (ATCC-TIB-15) were cultured in RPMI media (Fisher Scientific, Catalogue # A1049101) with 10% Fetal Bovine Serum (R&D Systems, Catalog # S12450H) and 1% Antibiotic-antimicotic (Thermofisher Scientific, Catalogue #15240062) up to a density of 1 million cells per mL prior to transfection.

Techniques: Activity Assay, Mutagenesis, Sequencing, Infection

Journal: bioRxiv

Article Title: Atlas of HIV cis-regulatory elements reveals extensive transcriptional variation across clades, isolates, and within individuals

doi: 10.64898/2026.04.03.716403

Figure Lengend Snippet: (A) MPRA activity in Jurkat cells tiling through the LTRs of HIV-1 and HIV-2. (B, G) Saturation mutagenesis MPRA experiments in unstimulated Jurkat cells for tiles in the LTR (B), gag/env (G) regions of HIV-2 ROD strain that show transcriptional activity. Region coordinates are provided using standardized SIVmac239 genomic coordinates. TF motifs that contribute to activity are outlined. (C) Saturation mutagenesis MPRA experiments in unstimulated and stimulated Jurkat cells for tile SIVmac239:379-571 in the HIV-2 LTR. (D) Violin plots showing the distribution of LTR activity in unstimulated Jurkat cells across 41 HIV-2 isolates with full-length sequences in NCBI. The thick black line indicates the median, and the dotted lines indicate the first and third quartiles. The dots heatmaps below indicate the fold activation by stimulation of Jurkat cells with αCD3+PMA, TNFα, or IFNγ. The color indicates the median fold activation across isolates, whereas the size of the dots reflects the fold activity differences between the 5th and 95th percentiles. (E) CASCADE-derived motifs for different cofactors in the LTR and gag regions of HIV-2 ROD strain. (F) MPRA activity map across the genome of HIV-2 ROD strain in unstimulated Jurkat cells. The genome organization is shown below. (H-I) Los Alamos National Laboratory (LANL) genome alignments of HIV-2 strains using the SIVmac239 genome as a reference. (H) Transcriptional activity predicted using CREST is shown in shades of green. (I) Transcription start sites predicted using Puffin trained on FANTOM CAGE data are shown in shades of red.

Article Snippet: Jurkat cells (ATCC-TIB-15) were cultured in RPMI media (Fisher Scientific, Catalogue # A1049101) with 10% Fetal Bovine Serum (R&D Systems, Catalog # S12450H) and 1% Antibiotic-antimicotic (Thermofisher Scientific, Catalogue #15240062) up to a density of 1 million cells per mL prior to transfection.

Techniques: Activity Assay, Mutagenesis, Activation Assay, Derivative Assay

Journal: The Journal of Biological Chemistry

Article Title: Natural killer cells induce distinct modes of cancer cell death: Discrimination, quantification, and modulation of apoptosis, necrosis, and mixed forms

doi: 10.1074/jbc.RA118.004549

Figure Lengend Snippet: Viability and apoptosis analysis of Jurkat pCasper (Jurkat pC) and K562 pCasper (K562 pC) cells. a and b, viability of Jurkat pC and K562 pC cells was analyzed in 96-well plates. Cells were imaged every 5 min in the brightfield, FRET fluorescence of pCasper, and GFP fluorescence of pCasper with a ×20 objective over 6 h with the Cell Observer microscope under incubator conditions (37 °C, 5% CO2). Spontaneous signs of apoptosis are indicated by the reduction of the FRET signal and corresponding gain of the GFP signal (green color in the overlay of the brightfield, GFP, and FRET channels). Counting dead cells after 6 h revealed that in these conditions, >95% were viable. c and d, the same experimental setup as in a or b, except that 2.5 μm staurosporine or 5 μg/ml anti-CD95 antibody Apo 1-1 was applied at time 0. e, quantification of FRET signals. As in c, 2.5 μm staurosporine was applied to K562 pC cells at time 0. In addition to the brightfield, GFP, and FRET overlay as shown in d, FRET-Youvan and FRET donor ratio are calculated (both color-coded). Low values are shown in black to dark blue, and high ones are shown in red to white. R1–R4 are marked and turn apoptotic during the experiment. f, FRET donor ratio kinetics of the cells R1–R4 from e over 6 h. g, quantification of all cells from a–d. The time point of apoptosis induction in each cell was quantified by the abrupt reduction of the FRET donor ratio signal as shown in f. h, Jurkat cells were transfected with the Casper-GR construct with a DEVA mutation in the caspase-binding site. Overlay of brightfield, GFP, and FRET channel and the FRET donor ratio is depicted for two cells stimulated with 2.5 μm staurosporine or 5 μg/ml anti-CD95 antibody Apo 1-1. Both cells are clearly apoptotic after 2 h, as indicated by morphological changes but show no change in fluorescence signals or the FRET donor ratio (h, quantification of n = 10 cells per condition in i).

Article Snippet: 3 × 10 6 Jurkat E6-1 (ATCC, TIB-15 TM ) or K562 (ATCC 1994) were transfected with 2 μg of pCasper3-GR vector (Evrogen) ( 20 ) using a Nucleofector® II (Lonza, program C-016).

Techniques: Fluorescence, Microscopy, Transfection, Construct, Mutagenesis, Binding Assay

Journal: The Journal of Biological Chemistry

Article Title: Natural killer cells induce distinct modes of cancer cell death: Discrimination, quantification, and modulation of apoptosis, necrosis, and mixed forms

doi: 10.1074/jbc.RA118.004549

Figure Lengend Snippet: Quantification of target cell apoptosis and necrosis by pCasper induced by primary human NK cells. a, nonlabeled NK cells were applied to Jurkat pCasper target cells loaded in addition with Fura2-AM. Cells were imaged in AIMV medium with the Cell Observer every 10 s, and overlays of brightfield + Fura-2 (at 360 nm) or brightfield + GFP + FRET and the FRET donor ratio are shown over time. White arrows mark the two contact sites between one NK cell and two different target cells. b, average Fura-2, GFP, and FRET fluorescence of five target cells from three experiments with typical necrotic morphological changes (cell swelling, loss of cytosol and organelles), such as the one marked by the white arrow in a at time 0. c, average Fura-2, GFP, and FRET fluorescence of five target cells from three experiments with typical apoptotic morphological changes (cell shrinking, blebbing, stop of organelle movement), such as the one marked by the white arrow in a at time 5 min. d, calcein (500 nm)-loaded Jurkat E6-1 target cells were imaged with the Cell Observer in 0.5 mm Ca2+ Ringer's solution containing 100 μm propidium iodide. Time 0 was set to the start of fluorescence changes of 1%. One representative killing event is depicted. e, average fluorescence of seven cells from five experiments, such as the one shown in d. f, same as d except that calcein fluorescence is not shown, and propidium iodide is indicated by a color code. One representative killing event is depicted. g, relative fluorescence changes (F/F0) of the cell shown in f along the dotted line starting from the nonlabeled NK cell. Scale bars, 10 μm. Error bars, S.E.

Article Snippet: 3 × 10 6 Jurkat E6-1 (ATCC, TIB-15 TM ) or K562 (ATCC 1994) were transfected with 2 μg of pCasper3-GR vector (Evrogen) ( 20 ) using a Nucleofector® II (Lonza, program C-016).

Techniques: Fluorescence

Journal: The Journal of Biological Chemistry

Article Title: Natural killer cells induce distinct modes of cancer cell death: Discrimination, quantification, and modulation of apoptosis, necrosis, and mixed forms

doi: 10.1074/jbc.RA118.004549

Figure Lengend Snippet: NK cell contact–dependent target cell death plot. Jurkat pCasper cells can be used to distinguish target cell apoptosis, primary necrosis, secondary necrosis (after initial apoptosis), and a mixed phenotype (parallel apoptosis and necrosis) induced by NK cells. Cells are from seven experiments for a–h. a, example of target cell apoptosis (white arrow), as evident by the morphological changes (blebbing, cell shrinking, stop of organelle and cell movement). b, time-resolved averaged normalized GFP and FRET fluorescence and the calculated FRET donor ratio for 8 cells. c, example of target cell secondary necrosis following an earlier apoptosis, as evident from the increased GFP signal (white arrow) evident by the morphological changes (cell swelling, loss of cytosol, and organelles). d, time-resolved averaged normalized GFP and FRET fluorescence and the calculated FRET donor ratio for five cells. e, example of target cell primary necrosis (with no evident apoptosis; white arrow), as evident by the morphological changes (cell swelling and loss of cytosol and organelles). f, time-resolved averaged normalized GFP and FRET fluorescence and the calculated FRET donor ratio for six cells. g, example of target cell parallel apoptosis and necrosis (mixed phenotype, necrosis with caspase activity; white arrow), as evident by the morphological changes (blebbing and cell swelling) and fluorescence changes. h, time-resolved averaged normalized GFP and FRET fluorescence and the calculated FRET donor ratio for 5 cells. i–m, primary NK cells were left untreated or stimulated with 100 units/ml IL-2 for 24 h. Cells were settled in a 96-well plate and observed for 17 h with the high-content imaging microscope under incubator conditions (37 °C, 5% CO2). For each condition, six experiments (two experiments from each of three blood donors to exclude individual donor effects) were analyzed. The color code for target cells is as follows: viable (orange), apoptosis (green), secondary necrosis after apoptosis (dark gray), and primary necrosis including the mixed phenotype (light gray). Time point 0 marks the onset of apoptosis or primary/secondary necrosis, respectively. i, analysis of 40 NK cells with target cell contacts. j, 62 NK cells stimulated with 100 units/ml IL-2. k, 45 NK cells treated with 100 nm concanamycin A to inhibit necrosis. l, 52 NK cells treated with 20 μg/ml CD178 antibodies (two different clones) to inhibit apoptosis. m, 44 NK cells treated with 100 nm concanamycin A and 20 μg/ml CD178 antibodies (two different clones) to inhibit necrosis and apoptosis. NK to target cell ratio was 1:1, 10,000 each), about 62,000 cells/cm2. Scale bars, 10 μm. n, percentage of Jurkat pCasper target cells killed by primary necrosis after 17 h. o, percentage of Jurkat pCasper target cells killed by apoptosis and secondary necrosis after 17 h. p, percentage of total killing of Jurkat pCasper target cells after 17 h. Data are shown as mean ± S.D. (error bars) (n = 3 donors). p values were calculated using ordinary one-way analysis of variance. **; p < 0.01; ***, p < 0.001; ns, no significant difference.

Article Snippet: 3 × 10 6 Jurkat E6-1 (ATCC, TIB-15 TM ) or K562 (ATCC 1994) were transfected with 2 μg of pCasper3-GR vector (Evrogen) ( 20 ) using a Nucleofector® II (Lonza, program C-016).

Techniques: Fluorescence, Activity Assay, Imaging, Microscopy, Clone Assay

Journal: The Journal of Biological Chemistry

Article Title: Natural killer cells induce distinct modes of cancer cell death: Discrimination, quantification, and modulation of apoptosis, necrosis, and mixed forms

doi: 10.1074/jbc.RA118.004549

Figure Lengend Snippet: Induction of apoptosis and necrosis via purified perforin and granzyme B. Target cell death of Jurkat pCasper cells was analyzed following the addition of different combinations of recombinant perforin and granzyme B. a, 0 pg/μl perforin and 200 pg/μl granzyme B (63 cells); b, 50 pg/μl perforin and 200 pg/μl granzyme B (445 cells); c, 100 pg/μl perforin and 200 pg/μl granzyme B (516 cells); d, 200 pg/μl perforin and 200 pg/μl granzyme B (393 cells); e, 200 pg/μl perforin and 0 pg/μl granzyme B (167 cells). f, example with 0 pg/μl perforin and 1250 pg/μl granzyme B. Each experiment was carried out in a single well at incubator conditions (37 °C, 5% CO2) in Ca2+-free Ringer's. At −30 min, perforin and granzyme B were added, and at 0 min, 1.25 mm Ca2+ was added, which is required for perforin activity. Two time points and the target cell death plot of all cells are shown for each condition. Scale bars, 10 μm.

Article Snippet: 3 × 10 6 Jurkat E6-1 (ATCC, TIB-15 TM ) or K562 (ATCC 1994) were transfected with 2 μg of pCasper3-GR vector (Evrogen) ( 20 ) using a Nucleofector® II (Lonza, program C-016).

Techniques: Purification, Recombinant, Activity Assay

Journal: The Journal of Biological Chemistry

Article Title: Natural killer cells induce distinct modes of cancer cell death: Discrimination, quantification, and modulation of apoptosis, necrosis, and mixed forms

doi: 10.1074/jbc.RA118.004549

Figure Lengend Snippet: Ratio of apoptosis and necrosis in FasR-expressing compared with FasR-deficient target cells. 75.000 Jurkat pCasper (a and b) or K562 pCasper (c and d) target cells were settled in each well of a 96-well plate and observed for 17 h with the high-content imaging microscope under incubator conditions in AIMV medium (37 °C, 5% CO2) on a fibronectin-coated surface. NK cells (a and c) or NK cells incubated with 100 nm CMA (b and d) from the same blood donor were added at time 0. Target cell death following NK contact was monitored in the brightfield + GFP + FRET overlay and quantified as shown in Fig. 3. For quantification, 213 (a), 236 (b), 88 (c), and 143 (d) cells were analyzed from four experiments. Scale bars, 10 μm.

Article Snippet: 3 × 10 6 Jurkat E6-1 (ATCC, TIB-15 TM ) or K562 (ATCC 1994) were transfected with 2 μg of pCasper3-GR vector (Evrogen) ( 20 ) using a Nucleofector® II (Lonza, program C-016).

Techniques: Expressing, Imaging, Microscopy, Incubation

Journal: The Journal of Biological Chemistry

Article Title: Natural killer cells induce distinct modes of cancer cell death: Discrimination, quantification, and modulation of apoptosis, necrosis, and mixed forms

doi: 10.1074/jbc.RA118.004549

Figure Lengend Snippet: Calcium influx regulates the apoptosis to necrosis ratio. a–d, Jurkat pCasper and K562 pCasper were settled on a fibronectin-coated coverslip in the presence (a and c) or absence (b and d) of external calcium. NK cells were added at time 0, and killing events were observed for 75 min via brightfield + GFP + FRET overlay. a, in the presence of external calcium, the FasR-positive target cells Jurkat pCasper cells were killed in a mixture of necrosis, apoptosis, and mixed forms via the cumulative effect of perforin, granzymes, and FasL/FasR. b, in the absence of calcium, Jurkat pCasper cells were only killed via apoptosis due to the perforin's inability to adhere at the target cell's membrane. The residual FasL/FasR pathway is able to induce apoptosis in the absence of external calcium. c, in the presence of external calcium, the FasR-deficient K562 pCasper cells are killed via necrosis, accompanied by some mixed forms caused by perforin-mediated granzymes. d, in the absence of external calcium, K562 pC cells are not killed at all, because perforin cannot attach to the K562's membrane; nor can apoptosis be induced due to the lack of FasR. e, during cytotoxicity experiments as outlined in a, the external Ca2+ concentration was varied over a wide range by adding EGTA or CaCl2 to AIMV. Apoptotic cells are visible in a green color. f, frequency of apoptotic and necrotic Jurkat pCasper cell killing as a function of the external free Ca2+ concentration taken from pictures as shown in e 2 h after incubation with NK cells. Scale bars, 10 μm.

Article Snippet: 3 × 10 6 Jurkat E6-1 (ATCC, TIB-15 TM ) or K562 (ATCC 1994) were transfected with 2 μg of pCasper3-GR vector (Evrogen) ( 20 ) using a Nucleofector® II (Lonza, program C-016).

Techniques: Membrane, Concentration Assay, Incubation

Journal: The Journal of Biological Chemistry

Article Title: Natural killer cells induce distinct modes of cancer cell death: Discrimination, quantification, and modulation of apoptosis, necrosis, and mixed forms

doi: 10.1074/jbc.RA118.004549

Figure Lengend Snippet: Single target cell death analysis in a 3D collagen matrix. a, overview of NK cells 3 h after starting the experiment (stained by LysoTracker Red; red) and Jurkat pCasper target cells in a collagen matrix imaged by light-sheet microscopy. Some targets are still viable as indicated by the orange fluorescence, some are killed by apoptosis as indicated by green fluorescence (loss of the FRET signal, compare b), and others are killed by necrosis, which can be detected by loss of fluorescence (compare c). b, example of an individual target cell in the collagen matrix killed by apoptosis following NK cell contact. Contact time between the target cell and NK cell was defined as 0 min. The loss of FRET fluorescence after 30 min is evident by the change from orange to green color. c, example of an individual target cell in the collagen matrix killed by necrosis following NK cell contact. Contact time between the target cell and NK cell was defined as 0 min. The loss of fluorescence after 5 min is evident. d and e, kinetics of GFP fluorescence, RFP fluorescence, FRET signals, and donor ratios of the cells shown in b and c. f, MCF-7 cells transfected with pCasper, grown into spheroids, and embedded in a collagen matrix together with NK cells, 1 h after start of gelation. Shown is a maximum intensity projection of pCasper-FRET in red, pCasper-GFP in green, and LysoTracker Red also in red. Most target cells express pCasper. Most cells are still viable, but some apoptotic cells can be identified. Some MCF-7 cells show a strong LysoTracker signal. g, NK cells at the border of the spheroid are migrating actively and are identified and marked by their LysoTracker signal (depicted in cyan). h, a representative killing event of an MCF-7 cell is depicted. After contact with the NK cell, the target undergoes shrinking and a change from red to green fluorescence, indicative of apoptosis.

Article Snippet: 3 × 10 6 Jurkat E6-1 (ATCC, TIB-15 TM ) or K562 (ATCC 1994) were transfected with 2 μg of pCasper3-GR vector (Evrogen) ( 20 ) using a Nucleofector® II (Lonza, program C-016).

Techniques: Staining, Microscopy, Fluorescence, Transfection