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: International Journal of Molecular Medicine

Article Title: m 6 A in adipose tissue inflammation: A novel regulator of obesity and metabolic diseases (Review)

doi: 10.3892/ijmm.2026.5795

Figure Lengend Snippet: Role of m 6 A in adipogenesis. Insufficient adipogenesis in adipose tissue leads to persistent, chronic inflammation. m 6 A modification plays a crucial role in all stages of adipogenesis, from commitment to terminal differentiation. During commitment, METTL3 promotes lipogenic differentiation in BMSCs by regulating the m 6 A levels of PTH1R and JAK1, whereas silencing METTL14 reduces the expression of SMAD1, inhibiting BMSC proliferation. During terminal differentiation, m 6 A regulates MCE and the transition to mature adipocytes. FTO influences key genes such as ATG5, ATG7 and JAK2, affecting autophagy, STAT3 phosphorylation and adipogenesis. FTO knockout increases the m 6 A levels of CCND1 and CDK2, blocking MCE. m 6 A, N6-methyladenine; METTL, methyltransferase-like; PTH1R, parathyroid hormone 1 receptor; JAK, Janus kinase; BMSC, bone marrow mesenchymal stem cell; MCE, mitotic clone amplification; FTO, Fat mass and obesity-associated protein; ATG, autophagy-related; STAT3, signal transducer and activator of transcription 3; CCND1, cyclin D1; CDK2, cyclin-dependent kinase 2; IGF2BP1, insulin-like growth factor 2 mRNA-binding protein 1; YTHDF2, YTH domain family 2.

Article Snippet: In addition, for mitotic clone amplification (MCE) in the early stage of terminal differentiation, the inhibition of FTO expression in 3T3-L1 cells leads to increased m 6 A methylation levels of cyclin D1 (CCND1) and cyclin-dependent kinase 2, the protein expression of which is reduced after recognition by YTHDF2, resulting in blockade of the MCE process and in turn the inhibition of lipogenesis ( ) ( ).

Techniques: Modification, Expressing, Phospho-proteomics, Knock-Out, Blocking Assay, Amplification, Binding Assay

Journal: International Journal of Molecular Medicine

Article Title: m 6 A in adipose tissue inflammation: A novel regulator of obesity and metabolic diseases (Review)

doi: 10.3892/ijmm.2026.5795

Figure Lengend Snippet: Role of m 6 A in ATMs. ATMs are deeply involved in adipose tissue inflammation, and m 6 A plays critical roles in macrophage biology, including their development, activation, pyroptosis and metabolism of lipids. (A) m 6 A regulates macrophage development by targeting genes such as CCND1 and ATRX via YTHDF3, ALKBH5 and METTL3, affecting haematopoietic stem and progenitor cell differentiation. (B) m 6 A modification mediated by METTL3, METTL14 and IGF2BP2 controls macrophage activation and polarization by influencing key genes such as SPRED2, MYD88 and STAT1, which impact the NF-κB and PPAR-γ pathways. (C) m 6 A regulates macrophage pyroptosis by targeting CASPASE-1, IL-1β and MALAT1 and modulating pathways such as the PTBP1/USP8/TAK1 pathway. (D) Additionally, m 6 A affects macrophage lipid metabolism by regulating lipid uptake and cholesterol efflux through MSR1 and SR-B1. m 6 A, N6-methyladenine; ATMs, adipose tissue macrophages; CCND1, cyclin D1; ATRX, α-thalassemia X-linked intellectual disability syndrome; YTHDF3, YTH domain family 3; ALKBH5, alkB homologue 5; METTL, methyltransferase-like; IGF2BP2, insulin-like growth factor 2 mRNA-binding protein 2; SPRED2, sprouty-related EVH1 domain-2; MYD88, myeloid differentiation primary response 88; STAT1, signal transducer and activator of transcription 1; NF-κB, nuclear factor-κB; PPAR-γ, peroxisome proliferator-activated receptor γ; CASPASE-1, cysteinyl aspartate specific proteinase-1; IL, interleukin; MALAT1, metastasis-associated lung adenocarcinoma transcript 1; PTBP1, polypyrimidine tract-binding protein 1; USP8, ubiquitin-specific peptidase 8; TAK1, TGFβ-activated kinase 1; MSR1, macrophage scavenger receptor 1; SR-B1, scavenger receptor type B1; ROS, reactive oxygen species; TSC1, tuberous sclerosis complex 1; SOCS2, suppressor of cytokine signalling 2; GSDMD-N, gasdermin D N-terminal domain; OxLDL, oxidized low-density lipoprotein; MSR1, macrophage scavenger receptor 1; DDX5, DEAD-box helicase 5; MEHP, mono(2-ethylhexyl) phthalate.

Article Snippet: In addition, for mitotic clone amplification (MCE) in the early stage of terminal differentiation, the inhibition of FTO expression in 3T3-L1 cells leads to increased m 6 A methylation levels of cyclin D1 (CCND1) and cyclin-dependent kinase 2, the protein expression of which is reduced after recognition by YTHDF2, resulting in blockade of the MCE process and in turn the inhibition of lipogenesis ( ) ( ).

Techniques: Activation Assay, Cell Differentiation, Modification, Binding Assay, Ubiquitin Proteomics

Journal: Biochimica et biophysica acta. Molecular basis of disease

Article Title: Npc1 deficiency impairs microglia function via TREM2-mTOR signaling in Niemann-Pick disease type C.

doi: 10.1016/j.bbadis.2024.167478

Figure Lengend Snippet: Fig. 1. Loss of Npc1 impairs brain development in mouse. A Representative photograph of brain from Npc1−/−mice and littermate control at postnatal day 63 (P63). B Brain weight curves of Npc1−/−mice and littermate controls (n = 9; Page x genotype < 0.0001, Page < 0.0001, Pgenotype < 0.0001). C Representative coronal sections of Npc1−/−mice and littermate controls at P42 visualized by hematoxylin and eosin staining. D Quantification of the cortical thickness of mice from P1 to P63 (n = 3). E, F Representative confocal images and quantification of Filipin (white) and DAPI (blue) staining in the cortex of Npc1+/+ and Npc1−/−mice at P42 (n = 3). G, H Representative confocal images and quantification of GFAP (red) and DAPI (blue) staining in the cortex of Npc1+/+ and Npc1−/−mice at P42 (n = 3). I, J Repre sentative confocal images and quantification of MBP (green) and DAPI (blue) staining in the cortex of Npc1+/+ and Npc1−/−mice at P42 (n = 3). K, L Representative confocal images and quantification of cleaved-Caspase3 (c.CASP3, green) staining in the cortex of Npc1+/+ and Npc1−/−mice at P42 (n = 3). M Western blot analysis for c.CASP3, BAX and BCL2 protein expression in the brain of Npc1−/−mice and littermate controls (n = 3). Results are presented as Mean ± SEM. n.s., not sig nificant. *P < 0.05, **P < 0.01 and ***P < 0.001. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: The primary and secondary antibodies were used as follows: cleaved-Caspase3 (Cell Signaling Technology, USA; Cat No. 9664), BAX (Cell Signaling Technology, USA; Cat No. 14796), BCL2 (BOSTER, China; Cat No. BA0412), APOE (Abcam, UK; Cat No. ab183597), CTSD (Abcam, UK; Cat No. ab75852), Npc1 (Abcam, UK; Cat No. ab134113), Npc2 (Abcam, UK; Cat No. ab218192), TREM2 (Cell Signaling Technology, USA; Cat No. 76765), TREM2 (R&D Systems, USA; Cat No. AF1729), TREM2 (Abcam, UK; Cat No. ab305103), SYK (Cell Signaling Technology, USA; Cat No.13198), p-SYK (Cell Signaling Technology, USA; Cat No.2710), AKT (Cell Signaling Technology, USA; Cat No.4691), p-AKT (Cell Signaling Technology, USA; Cat No.13038), GSK3β (Cell Signaling Technology, USA; Cat No. 12456), p-GSK3β (Cell Signaling Technology, USA; Cat No. 5558), mTOR (Cell Signaling Technology, USA; Cat No. 2983), p-mTOR (Cell Signaling Technology, USA; Cat No. 5536), S6K1 (Cell Signaling Technology, USA; Cat No. 34475), p-S6K1 (Cell Signaling Technology, USA; Cat No. 9234), 4EBP1 (Cell Signaling Technology, USA; Cat No. 9644), p-4EBP1 (Cell Signaling Technology, USA; Cat No. 9451), CD63 (Abcam, UK; Cat No. ab217345), GFAP (Cell Signaling Technology, USA; Cat No. 80788), β-Actin (Affinity Biosciences, USA; Cat No. AF7018), β-Tubulin (Absin, China; Cat No. abs830032), Goat anti-Rabbit IgG-HRP (Absin, China; Cat No. abs20040), Goat anti-Mouse IgG-HRP (Absin, China; Cat No. abs20039), Rabbit anti-Sheep IgG-HRP (Abcam, UK; Cat No. ab6747).

Techniques: Control, Staining, Western Blot, Expressing