Journal: bioRxiv

Article Title: Transcriptional rewiring of NF-κB by HBZ and Fra-2 drives inflammatory chemoresistance in Tax-negative adult T-cell leukemia/lymphoma

doi: 10.64898/2026.09.30.755641

Figure Lengend Snippet: ( A ) Bimolecular fluorescence complementation (BiFC) assay using the p65 and Fra-2 constructs shown in the top panel, showing interaction between NF-κB p65 and Fra-2 in HEK293T cells. Venus fluorescence (green) indicates protein-protein interaction in cell nuclei. Co-immunoprecipitation of p65-Flag and Fra-2-HA overexpressed in ( B ) HEK293T cells and ( C ) Jurkat cells. 48 h after transfection, NF-κB p65 or Fra-2 was immunoprecipitated with anti-Flag or anti-HA magnetic beads, respectively, and interactions were visualized by immunoblotting against the other interactant. ( D ) Co-immunoprecipitation of endogenous Fra-2 and p65 in ATL-2 cells. Fra-2 was immunoprecipitated with anti-Fra-2 antibody, and bound p65 was detected by immunoblotting.

Article Snippet: For co-precipitation of transiently expressed proteins, HEK293T cells were transfected with expression vectors encoding the HA-Fra2 with an equimolar ratio of a vector encoding a Flag-epitope tagged NF-kB P65 protein using PEIMax transfection reagent (Polysciences).

Techniques: Bimolecular Fluorescence Complementation Assay, Construct, Fluorescence, Co-Immunoprecipitation Assay, Transfection, Immunoprecipitation, Magnetic Beads, Western Blot

Journal: bioRxiv

Article Title: Transcriptional rewiring of NF-κB by HBZ and Fra-2 drives inflammatory chemoresistance in Tax-negative adult T-cell leukemia/lymphoma

doi: 10.64898/2026.09.30.755641

Figure Lengend Snippet: ( A ) Schematic representation of NF-κB p65 and Fra-2 truncation mutants used in this study. Rel homology domain (RHD) of p65 and bZIP domain of Fra-2 are indicated. ( B-C ) Quantification of BiFC assay between Fra-2 WT and p65 truncation mutants. HEK293T cells were co-transfected with pBiFC-VC-Fra-2, pBiFC-VN-p65, the p65 mutants, or the Fra-2 mutants. 48 h after transfection, Venus fluorescence was measured using a Spark 10M microplate reader (TECAN) (excitation 514 nm, emission 529 nm). ( D–E ) Co-immunoprecipitation assays confirming domain requirements for the Fra-2/p65 interaction. Data are representative of 4 independent experiments. Statistical analysis: one-way ANOVA with Dunnett’s multiple comparisons test (*p < 0.01, **p < 0.001, ***p < 0.0001).

Article Snippet: For co-precipitation of transiently expressed proteins, HEK293T cells were transfected with expression vectors encoding the HA-Fra2 with an equimolar ratio of a vector encoding a Flag-epitope tagged NF-kB P65 protein using PEIMax transfection reagent (Polysciences).

Techniques: Bimolecular Fluorescence Complementation Assay, Transfection, Fluorescence, Co-Immunoprecipitation Assay, Analysis

Journal: bioRxiv

Article Title: Transcriptional rewiring of NF-κB by HBZ and Fra-2 drives inflammatory chemoresistance in Tax-negative adult T-cell leukemia/lymphoma

doi: 10.64898/2026.09.30.755641

Figure Lengend Snippet: NF-κB reporter luciferase activity in ( A ) HEK293T and ( B ) Jurkat cells expressing p65 along with c-Fos or Fra-2. Cells were co-transfected with an NF-κB-luciferase reporter plasmid and the indicated proteins. 48 hours after transfection, luciferase activity was measured and normalized to an internal control. ( C ) Immunoblot confirming protein expression in Jurkat cells. ( D ) NF-κB reporter activity in the presence of HTLV-1 Tax. HEK293T cells were co-transfected with an NF-κB-luciferase reporter plasmid, p65, and with or without Tax. ( E ) Immunoblot of cytoplasmic (C) and nuclear (N) fractions showing p65 localization in the presence or absence of Fra-2. Sp1 served as a nuclear-fraction control. ( F ) Immunoblot confirming Fra-2 and p65 expression in total cell lysates. ( G ) NF-κB p65 DNA-binding activity in HEK293T cells transfected with Flag-p65 alone or with HA-Fra-2, measured using the TransAM NF-κB p65 ELISA. Data are representative of 4 independent experiments. Statistical analysis: one-way ANOVA with Dunnett’s multiple comparisons test (*p < 0.01, **p < 0.001).

Article Snippet: For co-precipitation of transiently expressed proteins, HEK293T cells were transfected with expression vectors encoding the HA-Fra2 with an equimolar ratio of a vector encoding a Flag-epitope tagged NF-kB P65 protein using PEIMax transfection reagent (Polysciences).

Techniques: Luciferase, Activity Assay, Expressing, Transfection, Plasmid Preparation, Control, Western Blot, Binding Assay, Enzyme-linked Immunosorbent Assay, Analysis

Journal: bioRxiv

Article Title: Transcriptional rewiring of NF-κB by HBZ and Fra-2 drives inflammatory chemoresistance in Tax-negative adult T-cell leukemia/lymphoma

doi: 10.64898/2026.09.30.755641

Figure Lengend Snippet: ( A ) NF-κB reporter luciferase activity in Jurkat cells expressing p65 and Fra-2 in the presence or absence of HBZ. Cells were co-transfected with an NF-κB-luciferase reporter plasmid, p65, JunD, and Fra-2 alone or in a dimer with p65, and in the presence or absence of HBZ-Myc. We measured luciferase activity 48 hours after transfection. Immunoblotting confirms expression of p65, Fra-2, JunD, and HBZ. Co-immunoprecipitation assays showing ( B ) Fra-2-HA/JunD-Flag and ( C ) Fra-2-HA/p65-Flag interactions in the presence or absence of HBZ-Myc. HEK293T cells were co-transfected with p65-Flag, JunD-Flag, and Fra-2-HA alone or with HBZ-Myc. 48 hours after transfection, JunD-Flag ( B ) or Fra-2-HA ( C ) was immunoprecipitated, and bound proteins were detected by immunoblotting. We quantified band intensities using ImageJ software and normalized to the corresponding actin band intensity. ( D ) 2D-multiplex plot showing the Fra-2-HA/JunD-Flag complex in the absence or presence of HBZ-Myc as analyzed by PICO assay and quantified in ( E ) using PIQuant software (p < 0.001, Student’s t-test). ( F-G ) 2D-multiplex plot and associated quantification showing the Fra-2-HA/p65-Flag complex in the presence or absence of HBZ. (p < 0.001, Student’s t-test). Data are representative of 3 independent experiments.

Article Snippet: For co-precipitation of transiently expressed proteins, HEK293T cells were transfected with expression vectors encoding the HA-Fra2 with an equimolar ratio of a vector encoding a Flag-epitope tagged NF-kB P65 protein using PEIMax transfection reagent (Polysciences).

Techniques: Luciferase, Activity Assay, Expressing, Transfection, Plasmid Preparation, Western Blot, Co-Immunoprecipitation Assay, Immunoprecipitation, Software, Multiplex Assay

Journal: bioRxiv

Article Title: Transcriptional rewiring of NF-κB by HBZ and Fra-2 drives inflammatory chemoresistance in Tax-negative adult T-cell leukemia/lymphoma

doi: 10.64898/2026.09.30.755641

Figure Lengend Snippet: Promoter activity of ( A ) IL-1β, ( B ) IL-2, ( C ) IL-4, ( D ) IL-17, and ( E ) CSF2 (GM-CSF) measured by luciferase reporter assay. HEK293T cells were co-transfected with reporter luciferase promoter plasmids for the indicated cytokines along with HA-p65-Flag, Myc-Fra-2, or Mock. 48 hours after transfection, luciferase activity was measured. ( F ) Immunoblot confirming p65 and Fra-2 expression; actin served as a loading control. Data are representative of 4 independent experiments. Statistical analysis: one-way ANOVA with Dunnett’s multiple comparisons test (ns p > 0.05, *p < 0.01, **p < 0.001, ***p < 0.0001).

Article Snippet: For co-precipitation of transiently expressed proteins, HEK293T cells were transfected with expression vectors encoding the HA-Fra2 with an equimolar ratio of a vector encoding a Flag-epitope tagged NF-kB P65 protein using PEIMax transfection reagent (Polysciences).

Techniques: Activity Assay, Luciferase, Reporter Assay, Transfection, Western Blot, Expressing, Control, Analysis

Journal: bioRxiv

Article Title: Transcriptional rewiring of NF-κB by HBZ and Fra-2 drives inflammatory chemoresistance in Tax-negative adult T-cell leukemia/lymphoma

doi: 10.64898/2026.09.30.755641

Figure Lengend Snippet: ( A ) IL-1β promoter activity in HEK293T cells expressing Fra-2, p65, or both in the presence or absence of HBZ.. 48 hours after transfection, luciferase activity was measured (p < 0.0001, one-way ANOVA with Dunnett’s test). ( B ) Immunoblot confirming HBZ, p65, and Fra-2 expression; actin served as a loading control. ( C ) Chromatin immunoprecipitation (ChIP) showing Fra-2 enrichment at NF-κB and AP-1 binding sites within the IL1B, IL2, TNF, and CSF2 promoters in ATL-2 cells. Data are presented as fold enrichment relative to IgG control (*p < 0.01, **p < 0.001, two-way ANOVA). ( D–E ) ATL-2 cell viability after 4 hours of pre-treatment with Anakinra (10 µg/mL) followed by doxorubicin treatment for 24 hours. Cell viability was assessed using PrestoBlue vital dye and survival rate was calculated relative to DMSO controls. ( F–G ) Viability of CD8 + -depleted PBMCs from Type I ATL patients (n=4) after 4 hours of pre-treatment with anti-IL-1β antibody (clone 4M27, 1 µg/mL) followed by doxorubicin treatment for 24 hours. IC₅₀ values: 1.6 µM (Anakinra), 5.2 µM (anti-IL-1β) (p < 0.001, one-way ANOVA). Data are representative of 4 independent experiments.

Article Snippet: For co-precipitation of transiently expressed proteins, HEK293T cells were transfected with expression vectors encoding the HA-Fra2 with an equimolar ratio of a vector encoding a Flag-epitope tagged NF-kB P65 protein using PEIMax transfection reagent (Polysciences).

Techniques: Activity Assay, Expressing, Transfection, Luciferase, Western Blot, Control, Chromatin Immunoprecipitation, Binding Assay