hut78  (ATCC)

Journal: bioRxiv

Article Title: The pyruvate branch point controls lymphoid cancer cell dissemination

doi: 10.64898/2026.03.16.712182

Figure Lengend Snippet: Mitochondrial ROS promote migration and dissemination of malignant lymphocytes (A) Schematic representation of workflow of mROS sorting and subsequent transwell migration and xenograft studies. (B) Transwell migration of Jurkat cells sorted for five different mROS levels ranging from low to high as indicated in different colors. (C) Representative images of 18 FDG-PET/CT studies and hematoxylin and eosin (H&E)-stained liver sections of hepatic dissemination of sorted mROS lo , mROS bulk , and mROS hi Hut78 cells. Liver outlined by dotted white lines in PET/CT images and infiltrating lymphocyte aggerates outlined in yellow in H&E images (n=4 mice per group in PET/CT studies). (D) Quantification of hepatic infiltration of sorted Hut78 cells in H&E sections shown as percent of liver section area (n=2 mice). (E) Quantification of organ infiltration after xenografting of HH cells. Hepatic spread was quantified by flow cytometry using anti-hCD45 antibodies (n=4 mice). (F) Hepatic infiltration after second-generation xenografting of isolated mROS lo and mROS hi xenograft tumor cells (n=3 mice/group). (G) Gene set enrichment analysis of FACS-sorted mROS lo and mROS hi Jurkat cells. (H) Effect of antioxidants, N-acetylcysteine (NAC) and mitoTEMPO (mitoT), on migration of Jurkat cells sorted for different mROS levels ranging from low to high. (I) Effect of N-acetylcysteine (NAC) and mitoTEMPO (mitoT) on transwell migration of Hut78 cells and freshly isolated leukemic cells from patients with CTCL (n=4), ALL (n=14), and CLL (n=11). (J) Hepatic dissemination of Hut78 cells pretreated with NAC and mitoT. Hepatic spread was quantified by flow cytometry using anti-hCD45 antibodies (n=3 mice/group). (K) Effect of mitoTEMPO on ex vivo migration of xenograft cells isolated from subcutaneous primary tumors (SQ CD45+) and liver infiltrates (Liver CD45+). (L) Effect of antioxidant mitoTEMPO on ex vivo migration of freshly isolated CD3+ and CD19+ cells from CLL patients (n=6) and PBMC from healthy subjects (n=10). (M) Levels of mROS and transwell migration of Jurkat cells treated with increasing concentrations of phenformin (0, 1, 2, 4, 6, 10 uM), rotenone (0, 10, 50, 100, 200, 500 nM), and antimycin A (0, 25, 50, 100, 200, 400 nM) for 24 hours. (N) Effect of antioxidant mitoTEMPO on transwell migration of Jurkat cells treated with low (+) and high (++) concentrations of each phenformin (4 and 10 uM), rotenone (50 and 500 nM), and antimycin A (100 and 500 nM). (O) Hepatic dissemination of Hut78 cells pretreated with phenformin. Hepatic spread was quantified by flow cytometry using anti-hCD45 antibodies (n=4 mice/group). All data presented as mean ± SD of at least triplicate measurements; ns, non-significant. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < .001, ∗∗∗∗p < 0.0001.

Article Snippet: The human malignant T-cell lines, Hut78, HH, Jurkat, and human malignant B-cell and myeloid lines Daudi, Toledo, Su-DHL-6, and HL-60 were obtained from the American Tissue Culture Collection (ATCC, Manassas, VA).

Techniques: Migration, Positron Emission Tomography-Computed Tomography, Staining, Flow Cytometry, Isolation, Ex Vivo

Journal: bioRxiv

Article Title: The pyruvate branch point controls lymphoid cancer cell dissemination

doi: 10.64898/2026.03.16.712182

Figure Lengend Snippet: Promotion of malignant lymphocyte spread by mROS is mediated through HIF-1a (A) Levels of HIF-1a in sorted mROS lo and mROS hi Hut78 cells detected by flow cytometry and immunoblotting. Immunoblots from nuclear extracts with antibody against H2A-Z used as loading control. (B) Flow-cytometric quantification of HIF-1a in mROS lo and mROS hi cells of indicated cell lines. HIF-1a levels shown in mean fluorescence intensity. (C) Increased HIF-1a levels in isolated leukemic cells from patients with CTCL (n=2), ALL (n=3), and CLL (n=3). (D and E) Effect of HIF-1a knockout in Hut78 cells on invasive potential in transwell assays (D) and hepatic infiltration in vivo (E; n=3 animals per group). (F) Effect of PX-478 on ex vivo migration of freshly isolated CD3+ and CD19+ cells from CLL patients (n=6) and PBMC from healthy subjects (n=10). (G and H) Immunoblots (G) and corresponding transwell migration assay (H) of Jurkat cells transfected with either wild-type HA-tagged HIF-1a (HIF WT ) or mutant HA-tagged-HIF-1a P402A/P564A (HIF mut ) followed by treatment with antioxidants, NAC or mitoTEMPO. LE, low exposure, HE, high exposure. (I and J) Immunoblots (I) and corresponding transwell migration assays (J) of Jurkat cells treated with PHD inhibitor, IOX2, and either NAC or mitoTEMPO. LE, low exposure, HE, high exposure. (K and L) Immunoblots (K) and corresponding e x vivo transwell migration assays (L) of leukemic cells treated with IOX2 and either NAC or mitoTEMPO after isolation from patients with CTCL (n=3), ALL (n=8), CLL (n=8). LE, low exposure, HE, high exposure. (M) Gene set enrichment analysis of Jurkat cells treated with PX-478 and sorted into mROS lo and mROS hi subpopulations. (N) Effect of hypoxia (3% O 2 ) versus normoxia on transwell migration of Jurkat cells treated with mitoTEMPO or PX-478. All data presented as mean ± SD of at least triplicate measurements; ns, non-significant. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < .001, ∗∗∗∗p < 0.0001.

Article Snippet: The human malignant T-cell lines, Hut78, HH, Jurkat, and human malignant B-cell and myeloid lines Daudi, Toledo, Su-DHL-6, and HL-60 were obtained from the American Tissue Culture Collection (ATCC, Manassas, VA).

Techniques: Flow Cytometry, Western Blot, Control, Fluorescence, Isolation, Knock-Out, In Vivo, Ex Vivo, Migration, Transwell Migration Assay, Transfection, Mutagenesis

Journal: bioRxiv

Article Title: The pyruvate branch point controls lymphoid cancer cell dissemination

doi: 10.64898/2026.03.16.712182

Figure Lengend Snippet: Glucose is an essential fuel for migration through activation of mROS/HIF-1a (A) Clustering heatmap of metabolomic profiles of sorted mROS lo and mROS hi Jurkat cells (n=3 independent samples analyzed in triplicates). (B) Ratio of mitochondrial oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) in isolated mROS lo and mROS hi cells. (C and D) Glucose uptake observed in mROS lo and mROS hi cells isolated from mouse xenograft tumors (C; n=3 tumors of each cell line) and isolated leukemic cells (D; ALL, n=3 patients; CLL, n=3 patients). (E) Effect of glucose-limiting conditions or inhibition of glycolysis with 2-deoxyglucose (2-DG) on transwell migration. Cells were incubated with different concentrations of glucose or 2-DG (0, 2.5, 5, 10, 20, 25 mM for both) during migration in transwell chambers. (F) Effect of glucose-limiting conditions on migration of isolated mROS lo and mROS hi cells. (G and H) Effect of long-term culture in glucose-limiting media on transwell migration (G). Transwell assays were performed after 30 days in glucose-limiting conditions with unchanged glucose concentration (-Glc) or freshly added glucose during the 24 h migration studies (+Glc; H) (I) Gene set enrichment analysis of Jurkat cells in glucose-free conditions. (J and K) Representative immunoblots (J) and corresponding transwell migration assay (K) of Jurkat cells transfected with either wild-type HA-tagged HIF-1a (HIF WT ) or mutant HA-tagged-HIF-1a P402A/P564A (HIF mut ) in media containing 3 mM of glucose or 25 mM of 2-DG. (L and M) Representative immunoblots (L) and corresponding ex vivo transwell migration assays (M) of isolated leukemic cells treated with PHD inhibitor IOX2 and NAC or mitoTEMPO (ALL, n=4 patients; CLL, n=4 patients). LE, low exposure, HE, high exposure. (N) Glucose uptake observed in control and HIF1a-KO Hut78 cells. mROS lo in light blue bars; mROS hi in orange bars in (B-D, F, N). All data presented as mean ± SD of at least triplicate measurements; ns, non-significant. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < .001, ∗∗∗∗p < 0.0001.

Article Snippet: The human malignant T-cell lines, Hut78, HH, Jurkat, and human malignant B-cell and myeloid lines Daudi, Toledo, Su-DHL-6, and HL-60 were obtained from the American Tissue Culture Collection (ATCC, Manassas, VA).

Techniques: Migration, Activation Assay, Metabolomic, Isolation, Inhibition, Incubation, Concentration Assay, Western Blot, Transwell Migration Assay, Transfection, Mutagenesis, Ex Vivo, Control

Journal: bioRxiv

Article Title: The pyruvate branch point controls lymphoid cancer cell dissemination

doi: 10.64898/2026.03.16.712182

Figure Lengend Snippet: Metabolic control of migratory potential at the pyruvate branch point (A) Transwell migration of cells supplemented with 2.5, 5, 10 mM pyruvate in glucose-free media for 24 hours. (B) Levels of mROS in response to pyruvate supplementation (10mM) in glucose-free media. (C) Immunoblots of HIF-1a in Jurkat cells after pyruvate supplementation in glucose-free media. (D and E) Effect of mitoTEMPO (D) and PX-478 (E) on transwell migration in glucose-free media with and without supplemented pyruvate. (F) Effect of pyruvate supplementation on ex vivo migration of freshly isolated CD3+ and CD19+ cells from CLL patients (n=6) and PMBCs from healthy subjects (n=10) in glucose-free media. (G) Transwell migration assay of cells transfected with mitochondrial pyruvate carrier 1 siRNA (siMPC1), monocarboxylate transporter 1 siRNA (siMCT1) or scrambled siRNA (siSCR). (H) Effect of mitoTEMPO (mitoT) and PX-478 on migration of cells transfected with mitochondrial pyruvate carrier 1 siRNA (siMPC1) or scrambled siRNA (siSCR). (I) Gene set enrichment analysis of Jurkat cells treated with AZD3965 compared to untreated control. (J and K) Immunoblots (J) and corresponding transwell migration assays (K) of Jurkat cells treated with PHD inhibitor IOX2 and Oxamate, DCA, or AZD3965. (L) Representative images of livers of mice treated with AZD3965 versus control and corresponding quantitation of hepatic disease infiltration in xenograft studies of Hut78 cells (n=4 animals per group). (M) Effect of AZD3965 on ex vivo migration of freshly isolated CD3+ and CD19+ cells from CLL patients (n=6) and PBMC from healthy subjects (n=13). (N) Schematic summary of oxidative versus glycolytic metabolic programming controlling dissemination potential of lymphoid cancer cells through redox signaling. All data presented as mean ± SD of at least triplicate measurements; ns, non-significant. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < .001, ∗∗∗∗p < 0.0001.

Article Snippet: The human malignant T-cell lines, Hut78, HH, Jurkat, and human malignant B-cell and myeloid lines Daudi, Toledo, Su-DHL-6, and HL-60 were obtained from the American Tissue Culture Collection (ATCC, Manassas, VA).

Techniques: Control, Migration, Western Blot, Ex Vivo, Isolation, Transwell Migration Assay, Transfection, Quantitation Assay

Journal: Nucleic Acids Research

Article Title: A viral noncoding RNA is a master regulator of gene expression that defines host cell identity and function

doi: 10.1093/nar/gkag472

Figure Lengend Snippet: HSUR1 reprograms host gene expression and promotes cell survival. ( A ) GO and KEGG pathway analyses showing selected categories significantly enriched among HSUR1 target mRNAs. ( B ) Flow cytometry of cj38637-WT and cj38637-ΔHSUR1 cells stained with Annexin V–Alexa Fluor 647 and propidium iodide; quantification from three independent experiments shown on the right. ( C ) Caspase-3/7 activity in WT and ΔHSUR1 cells measured by Caspase-Glo 3/7 assay after FasL treatment. ( D ) Same as in panel (C), using HuT78 cells transduced with lentiviral vectors expressing GFP (control) or HSUR1 and treated with vehicle or FasL. ( E ) Volcano plot of differentially expressed transcripts in cj38637-ΔHSUR1 versus cj38637-WT cells. Y axis shows two different scales (separated by a break), with significant adjusted P -values shown above the dashed lines. ( F ) Summary of changes in intron retention and alternative splice site usage. ( G ) Summary of differentially spliced cassette exons. NCE, noncoding transcript exon; 5′UTR, 5′ untranslated region exon; FCE, first coding exon; FS-CE, frameshifting internal coding exon; non-FS-CE, non-frameshifting internal coding exon; LCE, last coding exon. *** P < .001, **** P < .0001.

Article Snippet: Stably transduced firefly- and Renilla -luciferase-expressing HuT78 cells (see below) were grown in complete medium plus 8 μg/ml of puromycin (InvivoGen) and 10 μg/ml of blasticidin (InvivoGen), whereas stably transduced HuT78 and HeLa cells expressing WT or mutant versions of HSUR1 were grown in complete medium plus 10 or 8 μg/ml of puromycin, respectively.

Techniques: Gene Expression, Flow Cytometry, Staining, Activity Assay, Caspase-Glo Assay, Transduction, Expressing, Control

Journal: Nucleic Acids Research

Article Title: A viral noncoding RNA is a master regulator of gene expression that defines host cell identity and function

doi: 10.1093/nar/gkag472

Figure Lengend Snippet: Dual role of the HSUR1 ARE in RNA stability and target repression. ( A ) Northern blot showing HSUR1 levels in HeLa-H1 cells transfected with control siRNA or siRNAs targeting KHSRP, HuR, or hnRNP D. U1 serves as a loading control. Quantification of three independent experiments is shown on the right; U1 snRNA signal was used for normalization. ( B ) Sequence of the HSUR1 5′ end and miR-142-3p showing mutations disrupting the first (m1), second (m2), or third (m3) AUUUA motif. ( C ) Reporter assay using HuT78 cells expressing the luciferase-PPFIA1 3′UTR reporter and co-transfected with mCherry/V5-ZFP36L2 and WT or mutant HSUR1 constructs as in panel (B). ( D ) Co-immunoprecipitation of miRNAs with αSm antibodies from HeLa-H1, HeLa-H1m142, and HeLa-H1-AREm1–m3 cells transfected with miR-142-3p. Northern blot probed for miRNAs, HSUR1, and U1 (immunoprecipitation control). Input, 5%; αSm IP, 100%. ( E ) Enrichment of HSUR1 target and control mRNAs in KHSRP immunoprecipitates from cj38637-WT and cj38637-ΔHSUR1 cells. Western blots on the right show IP efficiency using normal rabbit serum control and αKHSRP antibodies (E2E2U). Input, 5%; IP, 100%. ( F ) Same as in panel (E) using V5 immunoprecipitates from HeLa cells co-transfected with mCherry/V5-ZFP36L2 and either control or miR-142-3p.

Article Snippet: Stably transduced firefly- and Renilla -luciferase-expressing HuT78 cells (see below) were grown in complete medium plus 8 μg/ml of puromycin (InvivoGen) and 10 μg/ml of blasticidin (InvivoGen), whereas stably transduced HuT78 and HeLa cells expressing WT or mutant versions of HSUR1 were grown in complete medium plus 10 or 8 μg/ml of puromycin, respectively.

Techniques: Northern Blot, Transfection, Control, Sequencing, Reporter Assay, Expressing, Luciferase, Mutagenesis, Construct, Immunoprecipitation, Western Blot

Journal: Nucleic Acids Research

Article Title: A viral noncoding RNA is a master regulator of gene expression that defines host cell identity and function

doi: 10.1093/nar/gkag472

Figure Lengend Snippet: HSUR1 determines cell identity and function. ( A ) Western blot of representative HSUR1 target proteins in cj38637-WT and cj38637-ΔHSUR1 cells; cofilin serves as a loading control. ( B ) IFN-γ production in cj38637-WT and cj38637-ΔHSUR1 cells detected by western blot after PMA/ionomycin stimulation in the presence of brefeldin A and monensin; quantification of three replicates shown at right. HDAC1 provides a loading control. ( C ) Predicted composition of resting (R) and active (A) immune cell types inferred from gene expression using CIBERSORTx. T-reg, regulatory T cell; γδ-T, gamma-delta T cell; Tfh, T follicular helper cell; DC, dendritic cell; MΦ, macrophage; NK, natural killer cell. ( D ) NK-like cytotoxic activity of WT and ΔHSUR1 cells toward MOLT-4 target cells measured by CytoTox-Glo assay. ( E ) Flow cytometry analysis of cj38637-WT and cj38637-ΔHSUR1 cells for the indicated surface markers. ( F ) Same as in panel (E) for the indicated markers in HuT78 cells (Control) or same cells transduced with a lentiviral vector expressing HSUR1. * P < .05, ** P < 0.01, **** P < .0001.

Article Snippet: Stably transduced firefly- and Renilla -luciferase-expressing HuT78 cells (see below) were grown in complete medium plus 8 μg/ml of puromycin (InvivoGen) and 10 μg/ml of blasticidin (InvivoGen), whereas stably transduced HuT78 and HeLa cells expressing WT or mutant versions of HSUR1 were grown in complete medium plus 10 or 8 μg/ml of puromycin, respectively.

Techniques: Western Blot, Control, Gene Expression, Activity Assay, Glo Assay, Flow Cytometry, Transduction, Plasmid Preparation, Expressing

Journal: Nucleic Acids Research

Article Title: A viral noncoding RNA is a master regulator of gene expression that defines host cell identity and function

doi: 10.1093/nar/gkag472

Figure Lengend Snippet: HSUR1 regulates target mRNAs through sequences defined by iRICC-seq. ( A ) Schematic of constructs used for stable expression of luciferase reporters and transient expression of HSUR1. Renilla luciferase was expressed from the spleen focus-forming virus promoter, and firefly luciferase from the thymidine kinase (TK) promoter, either without a 3′UTR (Control) or fused to partial 3′UTRs of HSUR1 target mRNAs. ( B ) Luciferase assays validating HSUR1 binding sites. Normalized luciferase levels in FACS-sorted, GFP-positive HuT78 cells stably expressing either control or 3′UTR-containing luciferase reporters after transient transfection with the indicated plasmids. The sizes of 3′UTR fragments (in base pairs) are indicated between parentheses. ( C ) Same as in panel (B) but using reporters containing individual HSUR1 binding sites. ( D-E ) Same as in panel (C) for HNRNPA2B1 (A2B1)-BS2mut (D) and JARID2-BS2mut (E) reporters. * P < .05; ** P < .01; *** P < .001.

Article Snippet: Stably transduced firefly- and Renilla -luciferase-expressing HuT78 cells (see below) were grown in complete medium plus 8 μg/ml of puromycin (InvivoGen) and 10 μg/ml of blasticidin (InvivoGen), whereas stably transduced HuT78 and HeLa cells expressing WT or mutant versions of HSUR1 were grown in complete medium plus 10 or 8 μg/ml of puromycin, respectively.

Techniques: Construct, Expressing, Luciferase, Virus, Control, Binding Assay, Stable Transfection, Transfection

Journal: Nucleic Acids Research

Article Title: A viral noncoding RNA is a master regulator of gene expression that defines host cell identity and function

doi: 10.1093/nar/gkag472

Figure Lengend Snippet: HSUR1 represses target mRNAs through their 3′UTRs. ( A ) Quantitative real-time polymerase chain reaction (qRT-PCR) analysis of representative HSUR1 target mRNAs bound only at the 3′UTR (ADAR, HNRNPA2B1, FOXN3, PIP4K2B, UBA2, ILRUN, and JARID2) and control mRNAs (RABEP1, ECPAS, and GAA) in cj38637-WT and cj38637-ΔHSUR1 cells. ( B ) Western blot of HSUR1 targets in cj38637-WT and cj38637-ΔHSUR1 cells. Cofilin provides a loading control. ( C ) As in panel (A) in cj319-WT cells transiently transfected with control or HSUR1 ASO. ( D ) As in panel (A) in HuT78 cells sorted by FACS for GFP after transient transfection with either a plasmid expressing GFP and the U1 snRNA promoter (Control) or a plasmid expressing GFP and HSUR1 under the U1 promoter (HSUR1). * P < .05; ** P < .01; *** P < .001; **** P < .0001.

Article Snippet: Stably transduced firefly- and Renilla -luciferase-expressing HuT78 cells (see below) were grown in complete medium plus 8 μg/ml of puromycin (InvivoGen) and 10 μg/ml of blasticidin (InvivoGen), whereas stably transduced HuT78 and HeLa cells expressing WT or mutant versions of HSUR1 were grown in complete medium plus 10 or 8 μg/ml of puromycin, respectively.

Techniques: Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Control, Western Blot, Transfection, Plasmid Preparation, Expressing