murine cell lines el4  (ATCC)

Journal: Cellular Oncology

Article Title: BCAA catabolism mediates POU2AF1 propionylation to enhance T-ALL development

doi: 10.1007/s13402-026-01201-w

Figure Lengend Snippet: POU2AF1 promotes T-ALL development via transcriptional activation of SLC7A11. ( A ) Western blot analysis of SLC7A11 protein levels in the EL4 cell lines. ( B ) The protein level of SLC7A11 was determined in WT and KO T-ALL cells by Western blotting ( n = 3). ( C ) POU2AF1 and SLC7A11 levels were measured in bone marrow cells from transplanted WT, Bcat1 -KO, Pou2af1 -overexpressing WT or Bcat1 -KO mice by Western blotting. ( D ) Luciferase reporter assay evaluating the transcriptional activation of Slc7a11 by Pou2af1 -WT and Pou2af1 -K5R ( n = 3). ( E ) ChIP assay showing the binding of Pou2af1 -WT and Pou2af1 -K5R to the Slc7a11 promoter region ( n = 3). ( F – G ) Representative flow cytometric analysis of GFP + mCherry + leukemia cells (The markers for the indication of leukemia cells were GFP + and mCherry + , which were the tags for the MSCV- Notch1 -IRES-GFP plasmid and MSCV-mCherry overexpression plasmid) in peripheral blood from recipient mice 3 weeks after transplantation with WT and KO T-ALL cells overexpressing Slc7a11 . ( H – I ) Representative images and quantification of the liver, spleen and thymus morphology in recipient mice ( n = 3). ( J ) Survival analysis of recipient mice ( n = 5). The data are presented as the means ± SDs. One-way ANOVA with Tukey’s multiple comparison test ( G ), log-rank test ( J ) and two-way ANOVA with Sidak’s multiple comparison test ( D , I ) were used for comparisons of statistical significance (*, P < 0.05; **, P < 0.01; and ***, P < 0.001)

Article Snippet: These viral particles were subsequently used to infect NOTCH-GFP+ bone marrow leukemia cells, human cell lines Jurkat and MOLT-4 (ATCC), murine cell lines EL4 and L1210 (ATCC), as well as primary T-ALL patient samples, followed by assessment of in vitro cell proliferation.

Techniques: Activation Assay, Western Blot, Luciferase, Reporter Assay, Binding Assay, Plasmid Preparation, Over Expression, Transplantation Assay, Comparison

Journal: Cellular Oncology

Article Title: BCAA catabolism mediates POU2AF1 propionylation to enhance T-ALL development

doi: 10.1007/s13402-026-01201-w

Figure Lengend Snippet: Targeting BCAT1 metabolism significantly impaired T-ALL progression. ( A – D ) Analysis of the proliferation of EL4 ( A ), L1210 ( B ), Jurkat ( C ), and MOLT-4 ( D ) cells treated in vitro with 10 mM gabapentin (n=4). ( E ) Representative flow cytometric analysis of peripheral blood at 3 weeks post-transplantation from T-ALL model mice following treatment with IgG, anti-PD-1 antibody, low-BCAA, or combined low-BCAA + anti-PD-1 antibody. ( F ) Quantification of the peripheral blood data shown in Panel E (n=5). ( G ) Overall survival analysis of treated mice (n=5). ( H ) Schematic model illustrating how BCAT1-driven branched-chain amino acid metabolism promotes T-ALL progression via Kpr-mediated activation of POU2AF1. The data are presented as the means ± SDs. One-way ANOVA with Tukey’s multiple comparison test ( F ), log-rank test ( G ), and two-way ANOVA with Sidak’s multiple comparison test (A–D) were used for comparisons of statistical significance (*, P <0.05; **, P <0.01; and ***, P <0.001)

Article Snippet: These viral particles were subsequently used to infect NOTCH-GFP+ bone marrow leukemia cells, human cell lines Jurkat and MOLT-4 (ATCC), murine cell lines EL4 and L1210 (ATCC), as well as primary T-ALL patient samples, followed by assessment of in vitro cell proliferation.

Techniques: In Vitro, Transplantation Assay, Activation Assay, Comparison

Journal: Nature Communications

Article Title: Aiolos restricts the generation of antigen-inexperienced, virtual memory CD8 + T cells in mice

doi: 10.1038/s41467-025-67540-8

Figure Lengend Snippet: Analysis of Eomes expression in CD8 + T cells isolated from the spleens of uninfected WT and Ikzf3 -/- mice. a Representative flow contour plots depicting expression of Eomes in WT and Ikzf3 -/- CD8 + T cells; and b respective bar graphs showing percent (%) of Eomes + and numbers of Eomes-expressing CD8 + T cells (# counts, normalized to 6 × 10 5 events). c Representative histogram overlay for Eomes expression and associated data showing differences in median fluorescence intensity (MFI) fold change relative to WT control. For ( a – c ), data shown for 4 independent experiments, n = 14/group, mean ± SEM, two-sided, unpaired Student’s t-test, ****p ≤ 0.0001. d Representative flow plots and e associated bar graphs showing percent (%) of CD122 + and numbers of CD122-expressing CD8 + T cells (#: counts, normalized to 6 × 10 5 events), Data shown for 3 independent experiments, n = 11/group, mean ± SEM, two-sided, unpaired Student’s t-test, ****p ≤ 0.0001. f Analysis of publicly available Aiolos Chromatin Immunoprecipitation (ChIP)-Seq data (GSM5106065) and STAT5b ChIP-Seq data (GSM7887512) showing enrichment of Aiolos and STAT5b at the Eomes promoter region. Sequencing tracks were viewed using Integrative Genomics Viewer. The gene region cloned into a reporter vector for Eomes promoter activity is indicated. g , h EL4 T cells were transfected with an Eomes promoter-reporter construct in conjunction with vectors for Aiolos, Aiolos DNA binding mutant (Aiolos DBM) , constitutively active STAT5b (STAT5b CA ), or empty vector control. As a control for transfection efficiency, cells were concurrently transfected with SV40- Renilla , and luciferase activity was used as a readout for promoter activity. Luciferase promoter-reporter values were normalized to SV40- Renilla control and presented as relative to the empty vector. Immunoblot analysis of the indicated proteins confirming the overexpression of respective vectors. β-actin was used as a loading control. Data are representative of 4 independent experiments, n = 4/condition, mean ± SEM, one-way ANOVA with Tukey’s multiple comparisons test, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, and ****p ≤ 0.0001. Source data are provided as a file.

Article Snippet: The EL4 murine T cell lymphoma line (TIB-39) for transfection studies and YAC-1 cells (TIB-160) for cytotoxicity were acquired from the American Type Culture Collection (ATCC) and maintained in complete RPMI (RPMI media [catalog # 61870-036, Thermo Fisher Scientific] containing 10% FBS [catalog # 26140-079, Life Technologies] and 1% penicillin/streptomycin [catalog # 15140-122, Life Technologies]).

Techniques: Expressing, Isolation, Fluorescence, Control, Chromatin Immunoprecipitation, ChIP-sequencing, Sequencing, Clone Assay, Plasmid Preparation, Activity Assay, Transfection, Construct, Binding Assay, Mutagenesis, Luciferase, Western Blot, Over Expression

Journal: Blood Advances

Article Title: Thyroid hormones contribute to JAK/STAT pathway abnormal activation, promoting T-cell lymphoma dissemination

doi: 10.1182/bloodadvances.2024015332

Figure Lengend Snippet: THs increase JAK/STAT pathway aberrant activation in TCL via integrin αvβ3. (A) Representative western blot results of p-STAT1 (Y701), p-STAT3 (Y705), and p-STAT5 (Y694) after 15 minutes of treatment with THs and in the presence or absence of the JAK1/2 inhibitor, Ruxo. (B) Representative western blot results of p-STATs levels after 15 minutes of treatment with THs and in the presence or absence of the integrin αvβ3 inhibitor, Cile. (C) mRNA levels of GATA3 , MMP2 , MMP9 , CCND1 , and CCR4 genes after 6-hour treatment. (D) Representative images of gelatin zymography of MMP2 and MMP9 activity measured in supernatants of TCL cells treated for 24 hours with THs, Cile, and the STAT3 inhibitor, Crypto. Ruxo = 1 μM, Cile = 3 μM, and Crypto = 1 μM for CUTLL1; 2.5 μM for OCI-LY13.2; 5 μM for OCI-LY12; and 1 μM for EL4 cells. THs include: T3 = 1 nM and T4 = 100 nM. ∗P < .05; ∗∗P < .01; ∗∗∗P < .001 vs Veh #P < .05; ##P < .01; ###P < .001; ####P < .0001 vs THs. Crypto, cryptotanshinone; THs, thyroid hormones; p-STAT1, phosphorylated STAT1; THC, thyroid hormones + Cile; THR, thyroid hormones + Ruxo.

Article Snippet: Murine TCL cell line EL4 was obtained from the American Type Culture Collection.

Techniques: Activation Assay, Western Blot, Zymography, Activity Assay

Journal: Blood Advances

Article Title: Thyroid hormones contribute to JAK/STAT pathway abnormal activation, promoting T-cell lymphoma dissemination

doi: 10.1182/bloodadvances.2024015332

Figure Lengend Snippet: Bex and Cile effects on tumor STATs phosphorylation and metalloproteinases activity. (A) Graphical representation of the in vivo TCL model and treatment schedule including Veh, Bex with levothyroxine replacement (BexT4+), Cile alone, or Bex with levothyroxine and Cile (BexT4+Cile). (B) p-STAT1 and p-STAT5 determination by flow cytometry in EL4 cells (gated) from tumors of the different groups. (C) Gelatin zymography quantification of MMP2 and MMP9 activities in the tumors. (D) Gelatin zymography quantification of MMP2 and MMP9 activities measured in supernatants of EL4 cells treated for 24 hours with Bex and Cile in the presence of physiological concentrations of THs (n = 5). Cile = 3 μM, Bex = 1.5 μM, T3 = 1 nM, T4 = 100 nM. ∗P < .05; ∗∗P < .01 ; ∗∗∗P < .001 vs Veh.

Article Snippet: Murine TCL cell line EL4 was obtained from the American Type Culture Collection.

Techniques: Phospho-proteomics, Activity Assay, In Vivo, Flow Cytometry, Zymography

Journal: Blood Advances

Article Title: Thyroid hormones contribute to JAK/STAT pathway abnormal activation, promoting T-cell lymphoma dissemination

doi: 10.1182/bloodadvances.2024015332

Figure Lengend Snippet: Proteomic analysis of the in vivo EL4 TCL model. (A) Schematic representation of the in vivo TCL model and the proteomic workflow used to obtain samples for proteomic analysis. Venn diagrams illustrate the total number of proteins identified in bulk tumors under respective treatments (n = 3 per group). (B-C) Venn diagrams of the EL4 tumor proteomics analysis displaying significantly upregulated and downregulated proteins in tumors from BexT4+Cile-treated mice compared with those from Veh-treated mice. (D) Heat map derived from the proteomic differential expression analysis. Only selected proteins of interest related to angiogenesis, cell migration, metabolism, immune response, cellular transport, and cell proliferation and survival that were identified as significantly upregulated or downregulated are shown. (E) TF enrichment analysis was performed using gene set enrichment analysis of the differentially expressed proteins among paired BexT4+Cile and Veh samples. The plot shows TF pathways that are significantly upregulated (red) or downregulated (blue) in BexT4+Cile vs Veh tumors (false discovery rate of <25% and P value < .05%). FC, fold change.

Article Snippet: Murine TCL cell line EL4 was obtained from the American Type Culture Collection.

Techniques: In Vivo, Derivative Assay, Quantitative Proteomics, Migration

Journal: Blood Advances

Article Title: Thyroid hormones contribute to JAK/STAT pathway abnormal activation, promoting T-cell lymphoma dissemination

doi: 10.1182/bloodadvances.2024015332

Figure Lengend Snippet: Bex and Cile combinatorial effect on TCL dissemination. (A) Graphical representation of the in vivo metastatic models and treatment schedule including Veh, bexarotene with levothyroxine replacement (BexT4+), Cile alone, or Bex with levothyroxine and Cile (BexT4+Cile). (B) Quantification of the number of experimental metastases in the liver and kidneys in the different groups in EL4 in vivo model. (C) Representative photographs of the livers and kidneys from the mice of the different treatments. (D) Quantification of the number of experimental metastases in the lungs of the different groups of treatment in the CUTLL1 in vivo model. (E) Representative photographs of lungs and kidneys from NOD-SCID (arrows point to CUTLL1 micronodules). (F) Representative photographs of hematoxylin and eosin–stained lung, kidney, and liver tissues (n = 5 per group).

Article Snippet: Murine TCL cell line EL4 was obtained from the American Type Culture Collection.

Techniques: In Vivo, Staining