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: Oncogene

Article Title: Transgenic expression of E2F3a causes DNA damage leading to ATM-dependent apoptosis.

doi: 10.1038/onc.2008.138

Figure Lengend Snippet: Figure 2 Overexpression of E2F3a activates ATM. (a) Western blot analysis was performed on epidermal lysates from nontrans- genic (lanes 1,2, 5 and 6) or K5 E2F3a transgenic (lanes 3, 4, 7 and 8) mice that were either wild-type (lanes 1–4) or null (lanes 5–8) for Atm. Antibodies specific for E2F3, phospho-ATM S1981 and b-tubulin were used as indicated. (b) Skin sections from wild-type, K5 E2F3a, Atm/ and K5 E2F3a Atm/ mice were immunohis- tochemically stained for the phosphorylated form of p53 (serine 18 in mouse). Positively stained epidermal keratinocytes were identi- fied microscopically and the average number per 10 mm of linear epidermis from at least three mice per group is presented. (c) Western blot analysis was performed on lysates from primary NHFs (lanes 1–3) or primary fibroblasts from an AT patient (lanes 4–6) infected with AdCMV empty vector (lanes 1 and 4), treated with etoposide as a positive control for DNA damage (lanes 2 and 5) or infected with AdE2F3a (lanes 3 and 6). Antibodies specific for E2F3, phospho-p53 S15, phospho-ATM S1981 and b-tubulin were used as indicated. ATM, ataxia telangiectasia mutated; NHFs, normal human fibroblasts. *indicates statistical significance at Po0.05.

Article Snippet: The following antibodies were used to detect the indicated protein: phospho-ATM S1981 (Rockland, Gilbertsville, PA, USA), E2F3 (Santa Cruz Biotechnology, C-18), b-tubulin (Santa Cruz Biotechnology, Santa Cruz, CA, USA, H-235), phospho-p53 S15 (Cell Signaling Technology), b-actin (Santa Cruz Biotechnology, H-2350).

Techniques: Over Expression, Western Blot, Transgenic Assay, Staining, Infection, Plasmid Preparation, Positive Control

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: 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: Non-coding RNA Research

Article Title: A novel enhancer RNA, Hmrhl, positively regulates its host gene, phkb, in chronic myelogenous leukemia

doi: 10.1016/j.ncrna.2019.08.001

Figure Lengend Snippet: Expression and coding potential analysis of Hmrhl. a. Quantitative real time PCR analysis of Hmrhl expression showed that it is expressed in all human tissues (Brain, Heart, Kidney, lung, liver, pancreas, spleen, thymus, small intestine, colon, skeletal muscle, testes, prostate, ovary, placenta, leukocyte, from left to right) examined. Lowest expression was found in skeletal muscle (SM) which was taken as control, the level of which was considered as 1 and all others were plotted in comparison to it. Highest expression was seen in spleen (spln) followed by pancreas (Pnc), testis (Tst) and other tissues. b. Northern blot detection of Hmrhl. Total RNA from HEK 293T and K562 cell lines were separated on agarose gel and subsequently hybridized with DIG labelled Hmrhl specific riboprobe to detect the transcript (i). In parallel, methylene blue staining was used to determine the size of HMRHL, using 28 S rRNA (5 kb) and 18s rRNA (1.9 kb) as reference (ii). Note that the size of Hmrhl is similar to that of 28s rRNA, revealing that Hmrhl is about 5 kb in size. c. Protein-coding potential as determined by Broad Institute's PhyloCSF data and visualized in UCSC Genome Browser, showing that Hmrhl has no coding potential. d. Circular phylogenetic tree built in iTOL (Interactive Tree of Life).

Article Snippet: Since Hmrhl locus exhibited enhancer properties in K562 Chronic Myelogenous Leukemia cells, we examined the expression profile of Hmrhl across various human cancers using a cancer specific cDNA panel (Origene, USA) by real time qPCR.

Techniques: Expressing, Real-time Polymerase Chain Reaction, Northern Blot, Agarose Gel Electrophoresis, Staining

Journal: Non-coding RNA Research

Article Title: A novel enhancer RNA, Hmrhl, positively regulates its host gene, phkb, in chronic myelogenous leukemia

doi: 10.1016/j.ncrna.2019.08.001

Figure Lengend Snippet: Hmrhl locus exhibits hallmarks of enhancer. a. ENCODE data visualized through Integrated Genome Viewer (IGV) for DNase hypersensitive sites, p300 binding, enhancer specific histone marks, H3K27Ac and H3K4Me1 and the promoter specific histone mark, H3K4Me3 at the 5′ end of Hmrhl, only in K562 but not in GM12878 cells. Note the two prominent peaks (red) for the enhancer mark H3K27Ac in K562. b-c. Chromatin immunoprecipitation with Ab8895 (anti-H3K4Me1 antibody) and Ab4729 (anti-H3K27Ac antibody) followed by qPCR in K562 cells. Note the enrichment of both the enhancer marks at the 5′ end of Hmrhl in the IP fraction as compared to input/PIS/gene desert region (GD), that serves as a negative control.

Article Snippet: Since Hmrhl locus exhibited enhancer properties in K562 Chronic Myelogenous Leukemia cells, we examined the expression profile of Hmrhl across various human cancers using a cancer specific cDNA panel (Origene, USA) by real time qPCR.

Techniques: Binding Assay, Chromatin Immunoprecipitation, Negative Control

Journal: Non-coding RNA Research

Article Title: A novel enhancer RNA, Hmrhl, positively regulates its host gene, phkb, in chronic myelogenous leukemia

doi: 10.1016/j.ncrna.2019.08.001

Figure Lengend Snippet: Hmrhl locus exhibits hallmarks of enhancer contd. a. Encode data shows the binding of various transcription and PolII at the 5′ end of Hmrhl. We have retained the H3K27Ac peaks in this figure also for a reference. b. Schematic for chromatin interaction analysis (ChiaPET data) for Hmrhl. The large purple-black peak representing histone marks on the extreme left denotes the promoter of phkb gene while the small purple peak at the far right represents the 5'end of Hmrhl. ChiaPET data shows the interaction of Hmrhl locus with phkb promoter, as represented by two black boxes (blue arrows) connected by a black line in b. The Hmrhl locus is expanded below in c , showing that this locus has enhancer properties only in K562 cell line (orange-yellow color), but not in other cell lines like GM12878, HepG2 or hESC. Genomic segments are colour coded by ENCODE as denoted in d , with red colour signifying active promoter ( phkb promoter at far left, black arrow in b ) while orange colour represents active enhancer at Hmrhl locus at far right (red arrow in b ).

Article Snippet: Since Hmrhl locus exhibited enhancer properties in K562 Chronic Myelogenous Leukemia cells, we examined the expression profile of Hmrhl across various human cancers using a cancer specific cDNA panel (Origene, USA) by real time qPCR.

Techniques: Binding Assay

Journal: Non-coding RNA Research

Article Title: A novel enhancer RNA, Hmrhl, positively regulates its host gene, phkb, in chronic myelogenous leukemia

doi: 10.1016/j.ncrna.2019.08.001

Figure Lengend Snippet: Hmrhl is differentially expressed in various cancers. a. Expression of Hmrhl in various normal and cancer samples as observed by qPCR. Note that Hmrhl is highly upregulated in several lymphoma samples (bracket) in comparison to normal range (arrow). In fact, of all cancers, the highest levels of Hmrhl are seen in some of the lymphoma samples. b-c. qPCR analysis of Hmrhl and PHKB expression showing that both are over expressed in K562 leukemia condition as compared to GM12878 normal lymphocytes.

Article Snippet: Since Hmrhl locus exhibited enhancer properties in K562 Chronic Myelogenous Leukemia cells, we examined the expression profile of Hmrhl across various human cancers using a cancer specific cDNA panel (Origene, USA) by real time qPCR.

Techniques: Expressing

Journal: Non-coding RNA Research

Article Title: A novel enhancer RNA, Hmrhl, positively regulates its host gene, phkb, in chronic myelogenous leukemia

doi: 10.1016/j.ncrna.2019.08.001

Figure Lengend Snippet: Hmrhl functions as enhancer RNA for phkb gene. a. Lucifaerase assay showing the intense signal of reporter activity in K562 cells with insert 3 cloned in enhancer vector. Note the low level of luciferase signal obtained with insert 2 both with promoter and enhancer vectors. b. siRNA (Sigma) mediated down-regulation of Hmrhl causes down-regulation of PHKB in K562 cells treated with Hmrhl specific siRNA pool as compared to control cells without transfection and cells treated with scrambled siRNA as negative control. c-d. Smart pool siRNA (Dharmacon) were used against the Hmrhl region to downregulate Hmrhl and subsequently expression level of PHKB gene were checked by qPCR in both K562 and GM12878 cell lines. Scrambled siRNA was used as a negative control. Note the down regulation of PHKB only in K562.

Article Snippet: Since Hmrhl locus exhibited enhancer properties in K562 Chronic Myelogenous Leukemia cells, we examined the expression profile of Hmrhl across various human cancers using a cancer specific cDNA panel (Origene, USA) by real time qPCR.

Techniques: Activity Assay, Clone Assay, Plasmid Preparation, Luciferase, Transfection, Negative Control, Expressing

Journal: Blood Cancer Journal

Article Title: Bruton’s tyrosine kinase inhibition re-sensitizes multidrug-resistant DLBCL tumors driven by BCL10 gain-of-function mutants to venetoclax

doi: 10.1038/s41408-025-01214-y

Figure Lengend Snippet: A Heatmap of differentially expressed genes in RIVA cells containing vector or BCL10 mutants, (FC ≥ 1.5x up or down) . B Venn diagram of the number of differentially expressed genes in BCL10 mutants compared to vector. C Top 10 upregulated (adjusted p value ≤ 0.001) Jensen compartment complexes using genes upregulated with a FC ≥ 2. D The top overlapping Hallmark gene set pathways upregulated (FDR q-value ≤ 0.25) in BCL10 mutants. E Western blot of RIVA and HBL1 cells induced with doxycycline for 24 h and probed as indicated. F TNFα ELISA assay on cell supernatant from RIVA, HBL1, and U2932 cells containing vector or BCL10 mutants. G Boxplot of gene expression levels of TNFRSF13B and USP2 of untreated DLBCL cases comparing mutant versus wildtype BCL10 cases.

Article Snippet: To more fully assess impact of BCL10 mutations on treatment resistance, we interrogated the 960-compound TargetMol Epigenetic Library.

Techniques: Plasmid Preparation, Western Blot, Enzyme-linked Immunosorbent Assay, Gene Expression, Mutagenesis

Journal: Blood Cancer Journal

Article Title: Bruton’s tyrosine kinase inhibition re-sensitizes multidrug-resistant DLBCL tumors driven by BCL10 gain-of-function mutants to venetoclax

doi: 10.1038/s41408-025-01214-y

Figure Lengend Snippet: A Gene ontology analysis of genes upregulated in BCL10 mutant RIVA cells showing top 10 of 49 significantly upregulated ontologies (adjusted p < 0.001). Ontologies that were completely overlapping in genes were combined. B GSEA “BIOCARTA_CYTOKINE_PATHWAY” for BCL10 mutants (q = 0.082 S136X, q = 0.176 for R58Q). C Venn diagram of cytokine genes upregulated in BCL10 mutants with array validations indicated. D Boxplot of gene expression levels of CCL22 and IL7 of untreated DLBCL cases comparing mutant versus wildtype BCL10 cases. E ELISA assays of IL6, IL7 and TNFβ on cells supernatant of RIVA and HBL1 cells induced for 24 (IL7 and TNFβ) or 72 h (IL6) with doxycycline, ** p < 0.01; *** p < 0.001; **** p < 0.0001.

Article Snippet: To more fully assess impact of BCL10 mutations on treatment resistance, we interrogated the 960-compound TargetMol Epigenetic Library.

Techniques: Mutagenesis, Gene Expression, Enzyme-linked Immunosorbent Assay

Journal: Blood Cancer Journal

Article Title: Bruton’s tyrosine kinase inhibition re-sensitizes multidrug-resistant DLBCL tumors driven by BCL10 gain-of-function mutants to venetoclax

doi: 10.1038/s41408-025-01214-y

Figure Lengend Snippet: A Genes that were upregulated (FC ≥ 2) in both mutants were analyzed for enrichment in regulation by transcription factors from the ENCODE project through the online transcription factor analysis tool ChIP-X Enrichment Analysis Version 3 (ChEA3). Sixteen transcription factors were significantly upregulated in BCL10 mutants (FDR ≤ 0.05). B Schematic of transcription factors identified to be upregulated in ( A ). C Boxplot of gene expression levels of BATF and IRF4 of untreated DLBCL cases comparing mutant versus wildtype BCL10 cases. D Western blot of RIVA and HBL1 cells induced with doxycycline for 24 h and probed as indicated. E Western blot of RIVA and U2932 cells induced with doxycycline for 24 h and probed as indicated. F ELISA assays in RIVA AND U2932 cell supernatant probing for CXCL10 (RIVA - S136X: p = 0.0046, R58Q: p = 0.0003, U2932- S136X: p = 0.0003, R58Q: p = 0.0028).

Article Snippet: To more fully assess impact of BCL10 mutations on treatment resistance, we interrogated the 960-compound TargetMol Epigenetic Library.

Techniques: Gene Expression, Mutagenesis, Western Blot, Enzyme-linked Immunosorbent Assay

Journal: Blood Cancer Journal

Article Title: Bruton’s tyrosine kinase inhibition re-sensitizes multidrug-resistant DLBCL tumors driven by BCL10 gain-of-function mutants to venetoclax

doi: 10.1038/s41408-025-01214-y

Figure Lengend Snippet: A Dose response viability assays of RIVA cells treated with pirtobrutinib (S136X: p = 0.033, R58Q: p = 0.019), acalabrutinib (S136X: p = 0.07, R58Q: p = 0.07), and ibrutinib (S136X: p = 0.023, R58Q: p = < 0.0001). B Epigenetic Library (TargetMol) on doxycycline induced RIVA cells. Hits are compounds that significantly inhibited cell viability and compounds that had an increase or decrease in viability two standard deviations from the mean of the difference between BCL10 S136X and vector after 72 h of 10 μM drug treatment. Targets in red highlight mutant-driven resistance, mutant-sensitive targets are in blue, and compounds that sensitized both vector and BCL10 S136X are in purple. C Schematic of hits from 4B. D Dose response viability assays of RIVA cells treated with selcidemstat (p=ns), tovorafenib (p = ns), ulixertinib (S136X: p = 0.0125, R58Q: p = 0.0103), JNKi (p = ns), duvelisib (S136X: p = 0.126, R58Q: p = 0.0264), idelalisib (S136X: p = ns, R58Q: p = 0.004), capivasertib (S136X: p = 0.0002, R58Q: p = 0.005), venetoclax (S136X: p = 0.008, R58Q: p = 0. 0.046), and AZD1208 (PIMi) (S136X: p = 0.019, R58Q: p = 0.040).

Article Snippet: To more fully assess impact of BCL10 mutations on treatment resistance, we interrogated the 960-compound TargetMol Epigenetic Library.

Techniques: Plasmid Preparation, Mutagenesis

Journal: Blood Cancer Journal

Article Title: Bruton’s tyrosine kinase inhibition re-sensitizes multidrug-resistant DLBCL tumors driven by BCL10 gain-of-function mutants to venetoclax

doi: 10.1038/s41408-025-01214-y

Figure Lengend Snippet: A Normalized counts from gene expression data for BCL2 family genes BCL2, BCL2L1 , and BCL2A1 in the BCL10 mutants. B Western blot of proteins implicated in venetoclax resistance including BCL-xL, BFL1, and BCL2 in doxycycline induced RIVA cells. C Transcript levels of doxycycline induced RIVA cells treated with DMSO or 5uM pirtobrutinib for 24 h: BCL2 (vector: p = 0.0325, R58Q: p = 0.005, S136X: p = 0.0006), BCL2L1 (vector: p=ns, R58Q: p = 0.0484, S136X: p = 0.0007), BCL2A1 (vector: p = 0.0087, R58Q: p = 0.0121, S136X: p = 0.0005). D Mitochondrial membrane potential flow cytometry assay in RIVA vector compared to RIVA R58Q and S136X at baseline. E Time course for BCL-xL, BFL1, and BCL2 in 5 μM pirtobrutinib treated RIVA cells. F Transcript levels of VDACs and TOMM22 after 24-h treatment with pirtobrutinib in RIVA cells. G Mitochondrial membrane potential flow cytometry assay in RIVA vector compared to RIVA R58Q and S136X after treatment with DMSO, pirtobrutinib, venetoclax or the combination for 2, 6 or 20 h. H Western blot of doxycycline induced RIVA cells treated with DMSO, venetoclax, pirtobrutinib or the combination for 24 h.

Article Snippet: To more fully assess impact of BCL10 mutations on treatment resistance, we interrogated the 960-compound TargetMol Epigenetic Library.

Techniques: Gene Expression, Western Blot, Plasmid Preparation, Membrane, Flow Cytometry

Journal: Blood Cancer Journal

Article Title: Bruton’s tyrosine kinase inhibition re-sensitizes multidrug-resistant DLBCL tumors driven by BCL10 gain-of-function mutants to venetoclax

doi: 10.1038/s41408-025-01214-y

Figure Lengend Snippet: A Upregulated transcription factor protein-protein interactions in genes upregulated by the BCL10 mutants probed through the Enrichr website ( p < 0.05). B Western blot showing phosphorylated STAT3 and total STAT3 in HBL1 and RIVA cells containing BCL10 mutants. C IL6 transcript levels of RIVA vector and mutants treated with pirtobrutinib for 24 h ( p < 0.0001). D IL6 cytokine levels in RIVA vector and mutants supernatant treated/untreated with 10uM pirtobrutinib for 24 h. E , F Western blot of doxycycline induced RIVA cells treated with DMSO, venetoclax, pirtobrutinib or the combination for 24 h. G Western Blot analysis of RIVA S136X cells treated with increasing concentrations of ruxolitinib with or without tocilizumab (IL6 inhibitor). H Western Blot analysis of RIVA S136X cells treated with increasing concentrations of ruxolitinib with or without the addition of TNFa. I IL6 transcript levels of RIVA S136X cells treated with TNFa alone or with ruxolitinib. J Schematic of TNFa / IL6/JAK/STAT signaling in BCL10 mutant cells. K Dose response viability assays of RIVA and HBL1 cells treated with pirtobrutinib (HBL1 clone1: p = 0.0027, HBL1 clone2: p = 0.0013, RIVA clone1: p = 0.0029, RIVA clone2: p < 0001) and ibrutinib (HBL1 clone1: p = 0.0401, HBL1 clone2: p = 0.0028, RIVA clone1: p = 0.3089, RIVA clone2: p < 0001), clones1 and 2 are the mutation (S136X) engineered at BCL10 gene locus. L Synergy assessments of venetoclax plus pirtobrutinib in endogenous BCL10 mutants (S136X) with reported synergy score.

Article Snippet: To more fully assess impact of BCL10 mutations on treatment resistance, we interrogated the 960-compound TargetMol Epigenetic Library.

Techniques: Protein-Protein interactions, Western Blot, Plasmid Preparation, Mutagenesis