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, Control, Comparison, 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, Comparison

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, Control, Transfection, Negative Control, Expressing

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: 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: Journal of ethnopharmacology

Article Title: Dehydrocorydaline attenuates myocardial ischemia-reperfusion injury via the FoXO signalling pathway: A multimodal study based on network pharmacology, molecular docking, and experimental study.

doi: 10.1016/j.jep.2024.118738

Figure Lengend Snippet: Fig. 5. Prediction and validation of DHC and its potential targets by molecular docking and IHC analysis. (A) Molecular docking of STAT3 and DHC. (B) Molecular docking of MDM2 and DHC. (C) Molecular docking of CDK2 and DHC. (D) Molecular docking of PLK1 and DHC. (E) Molecular docking of CCND1 and DHC. (F) Binding of DHC to CCND1 as determined through microscale thermophoresis (MST). (G) Binding of DHC to MDM2 as determined through MST. (H) Binding of DHC to CDK2 as determined through MST. (I) Relative protein levels of p-FOXO1A (n = 3). (J) Representative IHC images of p-FOXO1A, CCND1, and p-MDM2. (K) Relative protein levels of CCND1 (n = 3). (L) Relative protein levels of p-MDM2 (n = 3). *indicates a significant difference compared with the sham group, # indicates a significant difference compared with the MIRI group: *p < 0.05, **p < 0.01, #p < 0.05, ##p < 0.01. DHC: dehydrocorydaline, IHC: immunohistochemical, MIRI: myocardial ischemia-reperfusion injury.

Article Snippet: The microscale thermophoresis (MST) technique was performed to validate the interaction between CCND1 (#HY-P72119, MedChemExpress, Shanghai, China), CDK2 (#HY-P70014, MedChemExpress, Shanghai, China), MDM2 (#HY-P701593, MedChemExpress, Shanghai, China) and DHC(#HY-N0674, MedChemExpress, Shanghai, China).

Techniques: Biomarker Discovery, Binding Assay, Microscale Thermophoresis, Immunohistochemical staining

Journal: Journal of ethnopharmacology

Article Title: Dehydrocorydaline attenuates myocardial ischemia-reperfusion injury via the FoXO signalling pathway: A multimodal study based on network pharmacology, molecular docking, and experimental study.

doi: 10.1016/j.jep.2024.118738

Figure Lengend Snippet: Fig. 7. Effects of DHC on the expression of predicted targets in the in vitro model of H/R injury. (A) Representative fluorescence images of the TUNEL assay. Photographs were taken at × 400 magnification. (B) Percentage of TUNEL-positive cells in each group. (C) Western blot showing the protein expression of cleaved- caspase 3 and cleaved-caspase 8 in each group. (D) Western blot showing the protein expression of p-FOXO1A, FOXO1A, CCND1, p-MDM2, and MDM2 in each group. (E) Relative protein levels of cleaved-caspase 3 and cleaved-caspase 8 measured in western blots (n = 3). (F) Relative protein levels of p-FOXO1A, FOXO1A, and p- FOXO1A/FOXO1A measured in western blots (n = 3). (G) Relative protein levels of p-MDM2, MDM2, and p-MDM2/MDM2 measured in western blots (n = 3). (H) Relative protein levels of CCND1 measured in western blots (n = 3). * indicates a significant difference compared to the control group; # indicates a significant difference compared to the H/R model group: *p < 0.05, **p < 0.01, #p < 0.05, ##p < 0.01. DHC: dehydrocorydaline, H/R: hypoxia/reoxygenation, TUNEL: TdT- mediated dUTP-biotin nick end labelling.

Article Snippet: The microscale thermophoresis (MST) technique was performed to validate the interaction between CCND1 (#HY-P72119, MedChemExpress, Shanghai, China), CDK2 (#HY-P70014, MedChemExpress, Shanghai, China), MDM2 (#HY-P701593, MedChemExpress, Shanghai, China) and DHC(#HY-N0674, MedChemExpress, Shanghai, China).

Techniques: Expressing, In Vitro, Fluorescence, TUNEL Assay, Western Blot, Control

Journal: International Journal of Molecular Sciences

Article Title: A Dual Role for FADD in Human Precursor T-Cell Neoplasms

doi: 10.3390/ijms232315157

Figure Lengend Snippet: Pharmacological arrest at the G2/M stage of the cell cycle. The cell cycle of the FADD-expressing (FADD) and FADD-deficient (NEG) JURKAT cell lines was evaluated without any treatment (untreated) or after treatment with 100 nM etoposide, 50 ng/mL nocodazole, and 10 nM paclitaxel for 18 h. ( A ) Representative histograms of cells stained with propidium iodine showing DNA content distribution. Cell cycle phase distribution was analyzed by flow cytometry. ( B ) Bar chart of cell cycle distribution from six independent experiments. One-way ANOVA was used to test for statistical significance: * p ≤ 0.05; ** p ≤ 0.01. Error bars represent the standard error of the mean (SEM).

Article Snippet: Additionally, we corroborated that FADD expression of stable cell lines was equivalent to the FADD endogenous level of the parental clone JURKAT A3 (A3; ATCC, CRL-2570) ( ).

Techniques: Expressing, Staining, Flow Cytometry

Journal: International Journal of Molecular Sciences

Article Title: A Dual Role for FADD in Human Precursor T-Cell Neoplasms

doi: 10.3390/ijms232315157

Figure Lengend Snippet: Interactome analysis using DIA-MS confirms FADD participation in energy metabolism processes. ( A ) Workflow of co-Immunoprecipitation-Mass Spectrometry. Endogenous FADD protein was co-immunoprecipitated from FADD-expressing (FADD) and FADD-deficient (NEG) JURKAT cell lines using magnetic beads. Samples were trypsin-digested and injected into the mass spectrometer for data-independence acquisition. Finally, the raw data were analyzed with Spectronaut software and R. ( B ) STRING interaction map of significant proteins that interact with FADD in JURKAT cells compared to JURKAT-deficient FADD (FADD NEG). Proteins were clustered based on K-means into five clusters. The functional enrichment analysis and adjusted p -value (adj. p -value) are shown for the three main clusters. ( C ) Validation of DIABLO–FADD interaction. The input and immunoprecipitated (IP) fractions were separated by SDS-PAGE and blotted with antibodies against anti-FADD and -DIABLO. The densitometry values are shown below. ( D ) Schematic representation to show interactions of FADD with proteins involved in different biological processes. Under non-apoptotic conditions, FADD interacts with DIABLO/Smac, which hampers FADD function inducing apoptosis. FADD also interacts with several proteins involved in the energy metabolism.

Article Snippet: Additionally, we corroborated that FADD expression of stable cell lines was equivalent to the FADD endogenous level of the parental clone JURKAT A3 (A3; ATCC, CRL-2570) ( ).

Techniques: Immunoprecipitation, Mass Spectrometry, Expressing, Magnetic Beads, Injection, Software, Functional Assay, Biomarker Discovery, SDS Page