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

Journal: Cell reports

Article Title: DNA-PKcs controls the cytotoxic T cell response to cancer and transplant allograft through regulating LAT-dependent signaling

doi: 10.1016/j.celrep.2025.116796

Figure Lengend Snippet: (A) Subcellular fractionation of E6.1 Jurkat T cells shows increased DNA-PKcs phosphorylation at S2056 after 2 min of 5 μg/mL αCD3/CD28 TCR stimulation, which is reduced by the DNA-PKcs inhibitor NU7441 (5 μM). (B and C) (B) ImageJ quantification reveals a 3.5-fold increase in pDNA-PKcs band intensity in whole-cell extract and (C) a 4.8-fold increase in pDNA-PKcs band intensity in cytosolic extract, with one dot representing one experiment. Data are represented as mean ± SEM. (D) LSCM imaging at 63× magnification of E6.1 Jurkat T cells reveals that two minutes of αCD3/CD28 TCR stimulation (5 μg/mL) increases protein expression (green) in the cytosol and at the plasma membrane alongside F-Actin (red). (E) Quantification of pDNA-PKcs by mean fluorescence intensity (MFI) is shown for whole-cell pDNA-PKcs and the ratio of pDNA-PKcs outside the nucleus. Data are represented as mean ± SEM. (F) Among PIKK family members, DNA-PKcs, but not ATM or ATR, is activated in the cytosol following TCR stimulation with 5 μg/mL αCD3/CD28 or 100 nM doxorubicin (DNA damage-inducing reagent) for 2 min in E6.1 Jurkat T cells. (G) ImageJ quantification reveals a 3-fold increase in pDNA-PKcs band intensity following both TCR stimulation (CD3/28) and DNA damage (doxorubicin), with one dot representing one experiment. Data are represented as mean ± SEM. (H) LSCM imaging at 63× magnification of E6.1 Jurkat T cells reveals that two minutes of αCD3/CD28 TCR stimulation increases only pDNA-PKcs cytosolic presence, but not pATM or pATR. (I and J) (I) Quantified MFI values and (J) the cytosolic-to-whole-cell ratio of phosphorylated PIKKs, with each dot representing a single cell. Data are represented as mean ± SEM. Scale bars, 5 μm. Representative western blots and microscopy images from n = 3 independent experiments, with all cells within the field of view quantified on ICC. Statistical significance determined using one-factor ANOVA plus Tukey’s multiple comparisons (α = 0.05, * p < 0.05, ** p < 0.01, *** p < 0.005, and **** p < 0.0001).

Article Snippet: Jurkat E6.1 human T cell leukemia (male) and Raji B cells (male) were obtained from American Type Culture Collection (ATCC) with certification of analysis.

Techniques: Fractionation, Phospho-proteomics, Imaging, Expressing, Clinical Proteomics, Membrane, Fluorescence, Single Cell, Western Blot, Microscopy

Journal: Cell reports

Article Title: DNA-PKcs controls the cytotoxic T cell response to cancer and transplant allograft through regulating LAT-dependent signaling

doi: 10.1016/j.celrep.2025.116796

Figure Lengend Snippet: (A and B) (A) LSCM imaging at 63× magnification and (B) histogram MFI analysis of 2-min αCD3/CD28 TCR-stimulated Jurkat T cells identify areas of colocalization where pDNA-PKcs and LAT peaks overlap (*) and areas where peaks do not overlap (arrow). (C) LSCM imaging at 63× magnification of SEE-pulsed Raji B cells (blue) cocultured and conjugated with E6.1 Jurkat T cells shows pDNA-PKcs (green) colocalized with LAT (red) at the immune synapse (arrow). (D) LSCM immune synapses were quantified by MFI of pDNA-PKcs and LAT by drawing a quantifying line along the interface of Raji B cell and Jurkat T cell, each dot representing an area of immune synapse. Data are represented as mean ± SEM. (E) Co-immunoprecipitation (coIP) in Jurkat T cells shows that DNA-PKcs interacts with LAT following TCR stimulation. (F) TCR stimulation increases LAT pull-down by DNA-PKcs 7.5-fold, which is reduced by DNA-PKcs inhibitor NU7441 to 2.5-fold when quantified on ImageJ with one dot representing one coIP experiment. Data are represented as mean ± SEM. (G) LSCM imaging at 63× magnification of E6.1 Jurkat T cells demonstrates LAT (red) localization at the plasma membrane with pDNA-PKcs (green) upon TCR stimulation, which decreases with NU7441 (5 μM). Arrows identify areas of colocalization. (H) LSCM quantification reveals significant increases in MFI for pDNA-PKcs and LAT after TCR stimulation, along with colocalization events, which decrease upon DNA-PKcs inhibition with NU7441. Each dot represents a single quantified cell. Data are represented as mean ± SEM. (I and J) shRNA-mediated knockdown of DNA-PKcs (>70% reduction) reduces total DNA-PKcs expression on western blot. Data are represented as mean ± SEM. (K) LSCM imaging at 63× magnification shows that shRNA inhibition of DNA-PKcs attenuates LAT (red) localization at the plasma membrane after 2 min of 5 μg/mL αCD3/CD28 TCR stimulation. (L) Quantification of LSCM with each dot representing one shRNA-transfected cell. Data are represented as mean ± SEM. Scale bars, 5 μm. Representative images and western blots from n = 3 independent experiments with all cells quantified within the field of view on ICC. Statistical significance determined using one-factor ANOVA plus Tukey’s multiple comparisons (α = 0.05, ** p < 0.01, ** p < 0.005, and **** p < 0.0001).

Article Snippet: Jurkat E6.1 human T cell leukemia (male) and Raji B cells (male) were obtained from American Type Culture Collection (ATCC) with certification of analysis.

Techniques: Imaging, Immunoprecipitation, Clinical Proteomics, Membrane, Inhibition, shRNA, Knockdown, Expressing, Western Blot, Transfection

Journal: Cell reports

Article Title: DNA-PKcs controls the cytotoxic T cell response to cancer and transplant allograft through regulating LAT-dependent signaling

doi: 10.1016/j.celrep.2025.116796

Figure Lengend Snippet: E6.1 Jurkat T cells were stimulated with αCD3/CD28 for 15 min and then lysed with Golgi fractionation buffer. (A) Total and phosphorylated DNA-PKcs (pDNA-PKcs) are present in both the cis - and trans -Golgi fractions. Upon TCR stimulation, the secretory fraction exhibits an increase in pDNA-PKcs expression in the presence of LAT. (B) LSCM imaging at 63× magnification shows that LAT expression at the plasma membrane is attenuated by both DNA-PKcs inhibition (NU7441, 5 μM) and inhibition of secretory vesicle blebbing (brefeldin, 10 μg/mL). (C) Quantification of LSCM, with each dot representing one area of the plasma membrane. Data are represented as mean ± SEM. (D) LSCM imaging at 63× magnification shows that inhibition of DNA-PKcs with NU7441 (5 μM) prevents early TCR signaling markers like pLck (p-Y394) and CD3ζ from localizing to the plasma membrane in Jurkat T cells. (E) Quantification of LSCM with each dot representing one cell (LAT and CD3ζ) or an area of the plasma membrane (pLck). Data are represented as mean ± SEM. Scale bars, 5 μm. Representative images and blots from n = 3 independent experiments with all cells within the field of view quantified on ICC. One-factor ANOVA plus Tukey’s multiple comparisons was used to determine statistical significance (α = 0.05, **** p < 0.0001).

Article Snippet: Jurkat E6.1 human T cell leukemia (male) and Raji B cells (male) were obtained from American Type Culture Collection (ATCC) with certification of analysis.

Techniques: Fractionation, Expressing, Imaging, Clinical Proteomics, Membrane, Inhibition

Journal: bioRxiv

Article Title: Duration of Initial Viremia Modulates Functional Properties of HIV-specific T Cell Receptors

doi: 10.64898/2026.01.29.702605

Figure Lengend Snippet: Analysis of HIV-specific TCR avidity and functional affinity . ( A ) Estimated TCR-pHLA binding strength for individual clonotypes, calculated as the average ratio of bound tetramer (antigen, Ag) UMIs to CD3 UMIs. Statistical comparisons were performed using a non-parametric t-test. Each donor contribution is depicted with a different colour matched to the donor. ( B ). Representative FACs plot showing a monoclonal Jurkat TCR stained with a non-specific control tetramer (B*42:01-TL9) and the cognate tetramer (B*58:01-KW11). TCR-engineered T cells were co-cultured with HLA-B*58:01 monoallelic antigen-presenting cells pulsed with graded peptide concentrations (2μM to 0.125μM) at a 1:1 ratio for 18 hours. The histograms illustrate dose-dependent CD69 expression for 726_KW11-specific TCR3 clone. ( C and D ). Representative peptide dose-response curves for ( C ) IW9-specific TCRs and ( D ) KW11-specific TCRs derived from early-treated donor 726 and late-treated donor 309. TCR identifiers correspond to clonotypes with paired α and β-chains confirmed by both Illumina and Oxford nanopore sequencing. ( E and F ) Representative steady-state SPR measurements showing binding interactions between soluble HLA-B*58:01-IW9 pHLA complex and four IW9-specific TCRs, two from the early ART-treated donor 726 ( E ) and two from the late ART-treated donor 309 ( F ). Individual plots indicate donor TCR IDs and corresponding dissociation constants (K D )

Article Snippet: The concentrated lentivirus, along with 4 μg/mL of polybrene (Sigma-Aldrich, St Louis, MO), was then used to transduce a TCR-null Jurkat cell line (BPS Bioscience Cat. No: 78539).

Techniques: Functional Assay, Binding Assay, Staining, Control, Cell Culture, Expressing, Derivative Assay, Nanopore Sequencing

Journal: bioRxiv

Article Title: Duration of Initial Viremia Modulates Functional Properties of HIV-specific T Cell Receptors

doi: 10.64898/2026.01.29.702605

Figure Lengend Snippet: Normalized dose-response curves showing CD69 upregulation in monoclonal TCR-expressing Jurkat cells stimulated with IW9 (top) or KW11 (bottom) peptides. The Y-axis indicates the % of CD69 + Jurkat cells, and the X-axis represents peptide concentration. Each curve corresponds to an individual TCR, labeled by its respective ID. Red curves denote TCRs derived from early-treated donors, while blue curves represent TCRs from late-ART-treated donors. Peptide dose-response curves for each TCR were generated to estimate antigen sensitivity, with EC 50 serving as a quantitative proxy for TCR functional responsiveness.

Article Snippet: The concentrated lentivirus, along with 4 μg/mL of polybrene (Sigma-Aldrich, St Louis, MO), was then used to transduce a TCR-null Jurkat cell line (BPS Bioscience Cat. No: 78539).

Techniques: Expressing, Concentration Assay, Labeling, Derivative Assay, Generated, Functional Assay