Journal: Journal of Biological Chemistry

Article Title: Modulation of T Cell Cytokine Production by Interferon Regulatory Factor-4

doi: 10.1074/jbc.m205895200

Figure Lengend Snippet: FIG. 1. Early activation events in IRF-4-transfected cells. A, whole cell extracts were prepared from Jurkat cells stably transfected with either a control or an IRF-4 expression vector, electrophoresed on a 7% SDS-polyacrylamide gel, and then analyzed by Western blotting using an anti-IRF-4 antibody (upper panel). The blot was later stripped and reprobed with a -actin antibody (lower panel) to ensure for equal loading. Extracts from untransfected Jurkat cells and HUT 78 served, respectively, as negative and positive controls. B, Jurkat-transfected cells were either left unstimulated or were stimulated with PMA (50 ng/ml) and ionomycin (1 M) for 24 h. The cells were then harvested and stained with either a phycoerythrin-labeled anti-CD69 (upper panel) or a phycoerythrin-labeled anti-CD25 antibody (lower panel) and analyzed by flow cytometry. Filled histograms represent unstimulated cells, whereas empty histograms represent cells stimulated with PMA and ionomycin. Left panel, vector transfectants; right panel, IRF-4 transfec- tants. Not shown is staining with an isotype-matched control, which did not reveal any significant differences between control and IRF-4 transfectants.

Article Snippet: Cell Lines and Cultures—The Jurkat (human T cell leukemia) cell line was obtained from American Type Culture Collection (ATCC, Manassas, VA).

Techniques: Activation Assay, Transfection, Stable Transfection, Control, Expressing, Plasmid Preparation, Western Blot, Staining, Labeling, Flow Cytometry

Journal: Journal of Biological Chemistry

Article Title: Modulation of T Cell Cytokine Production by Interferon Regulatory Factor-4

doi: 10.1074/jbc.m205895200

Figure Lengend Snippet: FIG. 4. IRF-4 transactivates the human IL-2 and IL-4 promoters. Control and IRF-4 Jurkat-transfected cells were transiently transfected with a luciferase reporter construct driven either by the human IL-2 promoter (left panel) or the human IL-4 promoter (right panel). The transfected cells were equally split into two 2-ml aliquots and then incubated for 4 h in the presence or absence of PMA (50 ng/ml) and ionomycin (1 M). The data are presented relative to the activity of the reporter construct in unstimulated control cells, which was set to 1.0, as indicated in each experiment. Results show the mean S.E. of five (for the IL-2 promoter) and six (for the IL-4 promoter) independent experiments.

Article Snippet: Cell Lines and Cultures—The Jurkat (human T cell leukemia) cell line was obtained from American Type Culture Collection (ATCC, Manassas, VA).

Techniques: Control, Transfection, Luciferase, Construct, Incubation, Activity Assay

Journal: Journal of Biological Chemistry

Article Title: Modulation of T Cell Cytokine Production by Interferon Regulatory Factor-4

doi: 10.1074/jbc.m205895200

Figure Lengend Snippet: FIG. 6. IRF-4 can act as a transactivator of the P1-IRF element. Control and IRF-4 Jurkat cells were transfected with a luciferase re- porter construct driven by either an oligomerized P1-IRF wt or an oligomerized P1-IRFM3 element. The transfected cells were equally split into two 2-ml aliquots and then incubated for 4 h in the presence or absence of PMA (50 ng/ml) and ionomycin (1 M). The data are presented relative to the activity of the reporter construct in unstimu- lated control cells, which was set to 1.0, as indicated, in each experi- ment. Results show the mean S.E. of three independent experiments.

Article Snippet: Cell Lines and Cultures—The Jurkat (human T cell leukemia) cell line was obtained from American Type Culture Collection (ATCC, Manassas, VA).

Techniques: Control, Transfection, Luciferase, Construct, Incubation, Activity Assay

Journal: Journal of Biological Chemistry

Article Title: Modulation of T Cell Cytokine Production by Interferon Regulatory Factor-4

doi: 10.1074/jbc.m205895200

Figure Lengend Snippet: FIG. 7. IRF-4 cooperates with NFAT in driving T cell cytokine production. A, vector and IRF-4 Jurkat cells were co- transfected with a luciferase reporter con- struct driven by the human IL-4 promoter and either an NFATc1 expression vector or equivalent amounts of an empty vector. The transfected cells were equally split into two 2-ml aliquots and then incubated for 4 h in the presence or absence of PMA (50 ng/ml) and ionomycin (1 M). The data are presented relative to the activity of the reporter construct in vector control cells, which was set to 1.0, as indicated, in each experiment. Results show the mean S.E. of four independent experi- ments. B, control and IRF-4-transfected cells were either left unstimulated or stimulated with PMA and ionomycin as indicated in the legend to Fig. 2. Stimula- tions were conducted in the presence or absence of cyclosporin A (1 g/ml) or FK506 (10 ng/ml) as indicated. Superna- tants were then collected and analyzed for their cytokine content by ELISA. Data shown are representative of four inde- pendent experiments and performed on three independent sets of transfectants.

Article Snippet: Cell Lines and Cultures—The Jurkat (human T cell leukemia) cell line was obtained from American Type Culture Collection (ATCC, Manassas, VA).

Techniques: Plasmid Preparation, Transfection, Luciferase, Expressing, Incubation, Activity Assay, Construct, Control, Enzyme-linked Immunosorbent Assay

Journal: Nature Communications

Article Title: LANA-specific CD4 + effector T cells accumulate at the site of KSHV infection in humanized mice

doi: 10.1038/s41467-025-66992-2

Figure Lengend Snippet: A Degranulation assay measuring the frequency of CD107a + cells of CD4 + or CD8 + T cell clones upon co-culture with medium only (neg), PMA and Ionomycin (pos P/I), autologous unpulsed or cognate peptide pulsed LCLs or T2B35 cells. Mean ± SD of duplicates (Cl33/87/55/12/114pos) or triplicates (Cl156/114) of n = 1 experiment. Two-tailed unpaired t-test comparing unpulsed and pulsed conditions. B Schematic representation of the TCR construct used: variable α chain (Vα) followed by mouse constant α chain (mCα), P2A, variable ß chain (Vß), mouse constant ß chain (mCß), T2A, truncated mouse CD19 (mCD19). mCα and mCß are linked by an additional disulfide bond. Representative flow cytometry scatter plots of untransduced and transduced Jurkat Lucia NFAT cells showing the gating for transduced and TCR-expressing cells. C Jurkat Lucia NFAT reporter assay showing the fold change luminescent signal normalized to the background of unstimulated cells for transgenic TCR-transduced Jurkat Lucia NFAT cells stimulated with HLA-matched pulsed or unpulsed LCLs or T2B35 cells. Graph shows mean ± SD of n = 4 independent experiments. Two-tailed unpaired t-test. D Overview of the characteristics of each cloned TCR. Title contains information on the cognate antigenic peptide in brackets, the CDR3α and CDR3ß amino acid sequences, the TCR-Vß usage as well as the functional avidity EC 50 as determined in Fig. . Plot shows the HLA-restriction assessed via Jurkat Lucia NFAT reporter assays: fold difference of normalized luminescence signals of pulsed versus unpulsed LCLs or T2B35 cells is plotted for target cells sharing the same HLA-allotype (orange, x), target cells sharing the same HLA-allele group (light blue, HLA-allele group variants) and HLA-mismatched target cells (dark blue, line). The autologous LCL0 from the CD4 + T cell donor is DQB1*06:09, DRB1*13:02 and DRB3*03:01 positive. Graph shows mean ± SD of n = 1-5 independent experiments. Exact number n for each target cell tested is indicated subscripted in the y-axis labels. ( A–D ) * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. Exact significant p ≥ 0.0001 from left to right (1A) p = 0.0014, p = 0.0001; (1C) p = 0.0009, p = 0.0282, p = 0.0089, p = 0.0427.

Article Snippet: Jurkat-Lucia TM NFAT reporter cells (InvivoGen) were maintained in IMDM (Gibco, Thermo Fisher) supplemented with 10% FBS, 50 U/mL Penicillin/Streptomycin and 1% glutamine (I10).

Techniques: Degranulation Assay, Clone Assay, Co-Culture Assay, Two Tailed Test, Construct, Flow Cytometry, Expressing, Reporter Assay, Transgenic Assay, Functional Assay

Journal: Nature Communications

Article Title: LANA-specific CD4 + effector T cells accumulate at the site of KSHV infection in humanized mice

doi: 10.1038/s41467-025-66992-2

Figure Lengend Snippet: A Schematic of primary TCR-transduced T cell generation for downstream assays. B IFNɣ levels of primary TCR-transduced T cells or normalized luminescence of TCR-transduced Jurkat Lucia NFAT cells co-cultured overnight with HLA-matched LCLs pulsed with decreasing peptide concentrations. Graph shows mean ± SD of n = 2 (Jurkat Cl33), n = 3 (Jurkat Cl156/12, primary Cl114) or n = 4 (rest) independent experiments. C IFNɣ ELISA of TCR-transduced T cells co-cultured with cognate peptide-pulsed or unpulsed HLA-matched LCLs. Boxplots represent 5 (Cl114, Cl33), 4 (Cl12) or 3 (Cl156) independent experiments, using T cells from 8 (Cl114, Cl33, Cl12) or 6 (Cl156) different donors, tested against 9 (Cl114), 6 (Cl33), 4 (Cl156), 3 (Cl12) different LCLs. D Frequency of CD107a + T cells after 6 h of co-culture with cognate peptide-pulsed or unpulsed HLA-matching LCLs. Boxplots represent 3 (Cl114, Cl33, Cl12) or 1 (Cl156) independent experiments, using T cells from 6 (Cl12), 4 (Cl114, Cl33) or 2 (Cl156) different donors, tested against 6 (Cl114), 5 (Cl33), 4 (Cl156) or 3 (Cl12) different LCLs. E Specific killing of HLA-matched pulsed or unpulsed LCLs after co-culture with TCR-transduced T cells. Boxplots represent 3 (Cl156, Cl33, Cl12) or 2 (Cl114) independent experiments, using T cells from 6 (Cl156, Cl33), 5 (Cl12) or 3 (Cl114) different donors, tested against 5 (Cl114, Cl156, Cl33) or 2 (Cl12) different LCLs. C–E Paired two-tailed t-test. Intracellular cytokine staining (6 h) and surface marker assessment (18 h) of CD4 + ( F ) and CD8 + ( G ) T cells after stimulation with PMA/Ionomycin, cognate peptide-pulsed or unpulsed LCLs or medium only as a negative control. Mean frequency of cells positive for the indicated marker (y-axis) in each condition (x-axis) is plotted from 3 (CD4 + ) or 1 (CD8 + ) independent experiments with 5 (CD4 + ) or 2 (CD8 + ) different T cell donors. Numerical means are shown for LCL-stimulated conditions. Asterisks indicate significance from paired two-sided t-tests comparing pulsed versus unpulsed LCLs. A–E * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. Exact significant p ≥ 0.0001 from left to right (2D) 0.00015; (2E) p = 0.00236, p = 0.00019.

Article Snippet: Jurkat-Lucia TM NFAT reporter cells (InvivoGen) were maintained in IMDM (Gibco, Thermo Fisher) supplemented with 10% FBS, 50 U/mL Penicillin/Streptomycin and 1% glutamine (I10).

Techniques: Cell Culture, Enzyme-linked Immunosorbent Assay, Co-Culture Assay, Two Tailed Test, Staining, Marker, Negative Control

Journal: Nature

Article Title: Synthesis of Portimines Reveals the Basis of Their Anti-cancer Activity

doi: 10.1038/s41586-023-06535-1

Figure Lengend Snippet: a , Anti-proliferation activity of portimine A (PA) against human and mouse cancer cell lines (see Extended Data Fig. 1 for cell line descriptions). b , Structure of portimine related analogs: phenylportimine A (Ph-PA, 32 ) and epi-portimine A (ePA, 34 ), portimine A-diazirine-alkyne (PA-DA, 31-2 ) and epi-portimine A-diazirine-alkyne (ePA-DA, 31-1 ). c , Effects of PA and analogs on Jurkat cell viability (24 h; n = 3 biologically replicated experiments; mean ± s.d.). d , FACS analysis of apoptosis using annexin V/ eFluor 780 viability dye after PA and analogs treatment (12 h) in Jurkat cells ( n = 3 biologically independent samples; mean ± s.d.). Statistical analysis was performed using one-way ANOVA with multiple comparisons. P -values are shown. e , Immunoblot of caspase-3 and PARP1 after PA and analogs (12 h) treatment in Jurkat cells ( n = 3 biologically independent samples). f , PA induce G1-phase arrest. Jurkat cells were treated with portimine A or analogs for 12 h at 1 nM ( n = 3 biologically independent samples for each condition). Statistical analysis was performed for G1 phase distribution using one-way ANOVA multiple comparisons. P -values are shown. g , PA displays minimal toxicity in freshly isolated human PBMCs (36 h) from three different donors ( n = 3 biologically replicated experiments from each donor; mean ± s.d.). h-i , Impact of PA [0.3 or 1 mg kg −1 intraperitoneally] on h ) MC38 or i ) HT-1080 tumor growth in WT C57BL/6 mice ( n = 10 mice per group). Values indicate mean ± SEM. Mice were euthanized when tumor area exceeded 2000 mm 3 . Statistical analysis was performed using one-way ANOVA analysis with multiple comparisons. P -values are shown for day35 (MC38) or day21 (HT-1080) by comparing vehicle and 1 mg kg −1 PA treated group. For uncropped western blot images, see Supplementary Fig. 2 .

Article Snippet: For SAR experiments, Jurkat cells were plated at 50,000 cells per well in 96-well plates with media containing different concentrations of compounds in triplicate (0.2 pM – 1 μM) for 24 h. For caspase inhibitor rescue experiments, Jurkat cells were treated with media containing different concentrations of portimine A in triplicate (1, 5, 10 nM) with or without Z-VAD-FMK (50 μM, InvivoGen) in six replicates for 24 h. For cell screen experiments, cells were plated at 1,000 cells per well in 384-well plates with fresh media with different concentrations of portimine A in triplicate (8 pM – 500 nM) for 72 h. For Jurkat and PBMCs comparison experiments, cells were plated at 50,000 per well in 96-well plates with fresh media.

Techniques: Functional Assay, In Vivo, Activity Assay, Western Blot, Isolation

Journal: Nature

Article Title: Synthesis of Portimines Reveals the Basis of Their Anti-cancer Activity

doi: 10.1038/s41586-023-06535-1

Figure Lengend Snippet: Chemoproteomic profiling of PA in ( a ) Jurkat and ( b ) MC38 cells. X-axes correspond to PA-DA (500 nM) enriched proteins competed by PA (8 ×); y-axes correspond to proteins enriched by PA-DA over ePA-DA (500 nM). Designated PA-specific targets in red (competed by active competitor > 5-fold; enriched by PA-DA > 2-fold; and > 4-fold competition difference between PA and ePA). Dotted lines indicate competition (x-axis) and enrichment (y-axis) thresholds. Data presented as mean of biological replicates ( n = 2). See Supplementary Tables 7 - 9 for source data and Methods for additional details. c , Immunoblot of NMD3 engagement by PA-DA (500 nM) co-treated with PA or ePA (8 ×) as well as ePA-DA (500 nM). Representative of three biologically independent samples. d , Immunoblot of NMD3 thermal aggregation (CETSA) in the presence of PA (100 nM). Results representative of two biological replicates. e , Quantitation of the thermal aggregation curves ( n = 2 biological replicates; mean ± s.d.). f , Immunoblot of NMD3 in sh NMD3 Jurkat cells. g , PA (1 nM, 24 h) has reduced viability effects in Jurkat cells transduced with shRNA targeting NMD3 ( n = 6 biological replicates; mean ± s.d.). Statistical analysis performed using unpaired two-tailed Student t -test; P -values are shown. h , Polysome profiling of Jurkat cells treated with PA (50 nM, 6 h). Results representative of three independent experiments. i , Protein synthesis in Jurkat cells inhibited by PA, but not ePA (6 h), as measured by O -propargyl puromycin (OPP) incorporation. Data normalized to vehicle and presented as mean ± s.d. across biological replicates ( n = 3). Statistical analysis performed using multiple unpaired two-tailed Student t -test; P -values are shown. j , Immunoblot of Mcl-1 and c-Myc in Jurkat cells treated with PA (10 nM). Results representative of three biological replicates. For uncropped western blot images, see Supplementary Fig. 2 .

Article Snippet: For SAR experiments, Jurkat cells were plated at 50,000 cells per well in 96-well plates with media containing different concentrations of compounds in triplicate (0.2 pM – 1 μM) for 24 h. For caspase inhibitor rescue experiments, Jurkat cells were treated with media containing different concentrations of portimine A in triplicate (1, 5, 10 nM) with or without Z-VAD-FMK (50 μM, InvivoGen) in six replicates for 24 h. For cell screen experiments, cells were plated at 1,000 cells per well in 384-well plates with fresh media with different concentrations of portimine A in triplicate (8 pM – 500 nM) for 72 h. For Jurkat and PBMCs comparison experiments, cells were plated at 50,000 per well in 96-well plates with fresh media.

Techniques: Western Blot, Quantitation Assay, Transduction, shRNA, Two Tailed Test

Journal: Nature

Article Title: Synthesis of Portimines Reveals the Basis of Their Anti-cancer Activity

doi: 10.1038/s41586-023-06535-1

Figure Lengend Snippet: ( a ) PA induced-toxicity (24 h) can be rescued by caspase pan-inhibitor Z-VAD-FMK (50 μM) in Jurkat cells. Data are mean ± s.d. ( n = 6 biologically independent samples). Statistical analysis performed using multiple unpaired two-tailed Student t -test; P -values are shown. ( b-e ) Cell cycle analysis in Jurkat cells. Jurkat cells were treated with 1 nM of indicated compounds for 12 h and propodium iodide used to identify different stages of cells by flow cytometry. Shown are G1 ( b ), S ( c ), G2 ( d ) and SubG1 ( e ) phase distributions. Data are mean ± s.d of three independent biologically replicated experiments. Statistical analysis was performed using one-way ANOVA analysis with multiple comparisons. P -values are shown.

Article Snippet: For SAR experiments, Jurkat cells were plated at 50,000 cells per well in 96-well plates with media containing different concentrations of compounds in triplicate (0.2 pM – 1 μM) for 24 h. For caspase inhibitor rescue experiments, Jurkat cells were treated with media containing different concentrations of portimine A in triplicate (1, 5, 10 nM) with or without Z-VAD-FMK (50 μM, InvivoGen) in six replicates for 24 h. For cell screen experiments, cells were plated at 1,000 cells per well in 384-well plates with fresh media with different concentrations of portimine A in triplicate (8 pM – 500 nM) for 72 h. For Jurkat and PBMCs comparison experiments, cells were plated at 50,000 per well in 96-well plates with fresh media.

Techniques: Two Tailed Test, Cell Cycle Assay, Flow Cytometry

Journal: Nature

Article Title: Synthesis of Portimines Reveals the Basis of Their Anti-cancer Activity

doi: 10.1038/s41586-023-06535-1

Figure Lengend Snippet: ( a ) Chemical proteomic profiling with PA-DA in HCC1806. X-axis corresponds to PA-DA (500 nM) enriched proteins competed by PA (PA, 8 ×); y-axis corresponds to proteins enriched by PA-DA over ePA-DA (500 nM). Designated PA-specific targets in red (competed by active competitor > 5-fold; enriched by PA-DA > 2-fold; and > 4-fold competition difference between PA and ePA). Dotted lines indicate competition (x-axis) and enrichment (y-axis) thresholds. Data presented as mean of biological replicates ( n = 2). See Supplementary Tables 7 - 9 for source data. ( b-d ) Immunoblot of NMD3 engagement by PA-DA (500 nM) co-treated with PA or ePA (8 ×) as well as by ePA-DA (500 nM) in HeLa ( b ), HCC1806 ( c ), and MC38 cells ( d ). ( n = 2 biologically independent samples). ( e ) NMD3 is engaged by PA-DA in a dose-dependent manner in Jurkat cells. Results are representative of three independent experiments. ( f-i ) CETSA validation of NMD3 as a target of PA in Jurkat and MC38 cells. (f-g ) Immunoblotting and quantitation of NMD3 thermal aggregation curves (mean ± s.d.) in MC38 cells treated with PA. ( n = 3 biologically independent samples). ( h-i ) Dose-response (ITDR) fingerprint of NMD3 stabilization by PA in Jurkat cells and corresponding quantitation. Data presented as mean ± s.d. of biological replicated experiments ( n = 3). Statistical analysis performed using multiple unpaired two-tailed Student t -test; P -values are shown. ( j ) Isothermal dose-response (ITDR) fingerprint of NMD3 stabilization by PA in MC38 cells ( n = 3 biologically independent samples). For uncropped western blot images, see Supplementary Fig. 3 .

Article Snippet: For SAR experiments, Jurkat cells were plated at 50,000 cells per well in 96-well plates with media containing different concentrations of compounds in triplicate (0.2 pM – 1 μM) for 24 h. For caspase inhibitor rescue experiments, Jurkat cells were treated with media containing different concentrations of portimine A in triplicate (1, 5, 10 nM) with or without Z-VAD-FMK (50 μM, InvivoGen) in six replicates for 24 h. For cell screen experiments, cells were plated at 1,000 cells per well in 384-well plates with fresh media with different concentrations of portimine A in triplicate (8 pM – 500 nM) for 72 h. For Jurkat and PBMCs comparison experiments, cells were plated at 50,000 per well in 96-well plates with fresh media.

Techniques: Western Blot, Biomarker Discovery, Quantitation Assay, Two Tailed Test

Journal: Nature

Article Title: Synthesis of Portimines Reveals the Basis of Their Anti-cancer Activity

doi: 10.1038/s41586-023-06535-1

Figure Lengend Snippet: ( a ) Immunoblot of NMD3 in HeLa and MC38 cells expressing control or NMD3-specific shRNAs. ( b-c ) PA has reduced viability effects in Jurkat cells transduced with shRNA targeting NMD3 compared to shCtrl cells in HeLa ( b , 1 nM, 24 h) and MC38 ( c , 1 nM, 48 h). Data presented as mean ± s.d. of biologically replicated experiments ( n = 3 for HeLa, n = 6 for MC38). Statistical analysis was performed using unpaired two-tailed Student t -test, P -values are shown. ( d ) Quantitation of 60S:80S ratio and 80S:polysome ratio from data shown in Fig. 4f . Presented as mean ± s.d. of biological replicates ( n = 3). Statistical analysis performed using unpaired two-tailed Student t -test; P -values are shown. ( e ) Polysome profiling of MC38 cells treated with PA (50 nM, 6 h). Results are representative of two independent experiments. ( f ) PA, but not ePA, inhibits new protein synthesis in HeLa, HCC1806, and MC38 cells as determined by O -propargyl puromycin (OPP) incorporation. Results normalized to vehicle and presented as the mean ± s.d. across biologically replicated experiments ( n = 3). Statistical analysis was performed using multiple unpaired two-tailed Student t -test. P -values are shown. ( g, h ) Immunoblot of Mcl-1 and c-Myc after PA and ePA treatment in g ) Jurkat and h ) MC38 cells. Results are representative of two independent experiments. ( i ) Quantitation of protein abundances from Fig. 4j . Results presented as mean ± s.d. of biologically replicates ( n = 3). Statistical analysis was performed using multiple unpaired two-tailed Student t -test; P -values are shown. ( j ) MCL1 and MYC mRNA expression assessed by quantitative PCR in Jurkat cells treated with PA (10 nM). Results normalized to vehicle and values indicate mean ± s.d. ( n = 3 biologically independent samples). Statistical analysis performed using multiple unpaired two-tailed Student t -test. P -values are shown. For uncropped western blot images, see Supplementary Fig. 3 .

Article Snippet: For SAR experiments, Jurkat cells were plated at 50,000 cells per well in 96-well plates with media containing different concentrations of compounds in triplicate (0.2 pM – 1 μM) for 24 h. For caspase inhibitor rescue experiments, Jurkat cells were treated with media containing different concentrations of portimine A in triplicate (1, 5, 10 nM) with or without Z-VAD-FMK (50 μM, InvivoGen) in six replicates for 24 h. For cell screen experiments, cells were plated at 1,000 cells per well in 384-well plates with fresh media with different concentrations of portimine A in triplicate (8 pM – 500 nM) for 72 h. For Jurkat and PBMCs comparison experiments, cells were plated at 50,000 per well in 96-well plates with fresh media.

Techniques: Activity Assay, Western Blot, Expressing, Control, Transduction, shRNA, Two Tailed Test, Quantitation Assay, Real-time Polymerase Chain Reaction

Journal: iScience

Article Title: Amniotic epithelial Cell microvesicles uptake inhibits PBMCs and Jurkat cells activation by inducing mitochondria-dependent apoptosis

doi: 10.1016/j.isci.2025.111830

Figure Lengend Snippet: AEC MV cargo transfer promoted an immunosuppressive response in PBMCs and Jurkat cells (A) PHA-stimulated PBMCs ± Anti-CD44 and ± EIPA were treated with the different CM (AEC ± LPS MV and AEC± LPS MV-free ) for 48h and assessed for their proliferation. Data were normalized on PHA-stimulated PBMCs (100% of proliferation). (B) CD3/CD28-stimulated Jurkat reporter cells ± Anti-CD44 and ± EIPA were evaluated for the inhibition of NFAT activation after treatment with the different CM (±LPS) for 48h. Data were normalized on CD3/CD28 stimulated Jurkat (100% of NFAT activation). Data (mean ± SD) represent 3 independent sets of experiments (n = at least 3 biological replicates in each group per set; each biological replicate assayed in at least 3 technical replicates). ALL, ∗, ∗∗, ∗∗∗, and ∗∗∗∗ Statistically significant values between the different studied groups ( p < 0.01, p < 0.05, p < 0.01, p < 0.001, and p < 0.0001, respectively). One-way ANOVA was employed for comparing normally distributed data.

Article Snippet: Jurkat-Lucia TM NFAT cells , InvivoGen , Cat# jktl-nfat-cd28.

Techniques: Inhibition, Activation Assay

Journal: iScience

Article Title: Amniotic epithelial Cell microvesicles uptake inhibits PBMCs and Jurkat cells activation by inducing mitochondria-dependent apoptosis

doi: 10.1016/j.isci.2025.111830

Figure Lengend Snippet:

Article Snippet: Jurkat-Lucia TM NFAT cells , InvivoGen , Cat# jktl-nfat-cd28.

Techniques: Recombinant, Electrophoresis, Protease Inhibitor, Blocking Assay, Luciferase, Proliferation Assay, Flow Cytometry, Transgenic Assay, Software, Control

Journal: Developmental biology

Article Title: Fertilization stimulates 8-hydroxy-2'-deoxyguanosine repair and antioxidant activity to prevent mutagenesis in the embryo.

doi: 10.1016/j.ydbio.2015.07.024

Figure Lengend Snippet: Fig. 1. OGG1 is under-represented in MII-stage mouse oocytes. (A) Quantitative PCR analysis revealed a low abundance of OGG1 and APE1 mRNA within the murine oocyte (relative to B2M and Gusβ), whilst XRCC1 transcripts were present at significantly higher levels. (B) Immunoblotting analysis of the protein composition of these oocytes identified bands corresponding to APE1 and XRCC1 at 35 kDa and 85 kDa respectively; whilst a 37 kDa band corresponding to OGG1 could not be detected at this con- centration of protein (100 oocytes per lane, equivalent to 1 mg protein). Positive controls; rOGG1, rAPE1 and Jurkat cell lysate respectively (1 mg). (C, D) In support of Western blotting data, immunocytochemistry identified fluorescence associated with APE1 and XRCC1 throughout the ooplasm. Fluorescence relating to OGG1 was low, however upon pixel intensity analysis was still found to be significantly greater than the ‘secondary only’ control (“C”). Scale bar¼50 mm. Mean7SEM values are plotted in histograms. Independent replicates were conducted with a minimum of 40 oocytes per replicate. *Po0.05, **Po0.01, ***Po0.001.

Article Snippet: 1 mg of recombinant OGG1 (rOGG1), recombinant APE1 (rAPE1) (both from New England Biolabs, Ipswich, MA, USA), and Jurkat whole cell lysate (Novus Biologicals, Littleton, CO, USA) was loaded onto the polyacrylamide gels as a positive control for OGG1, APE1 and XRCC1 antibodies respectively.

Techniques: Real-time Polymerase Chain Reaction, Western Blot, Immunocytochemistry, Fluorescence, Control