Fig 1.
(A-B) Co-IP analysis with either Tax (A) or Flag-UBE4B (B) immunoprecipitates from lysates of 293T cells transfected with the indicated plasmids. (C-D) Co-IP analysis with either control IgG, anti-UBE4B, anti-NEMO or anti-Tax immunoprecipitates from lysates of C8166 (C) and MT-2 (D) cells as indicated.
Fig 2.
Co-localization of UBE4B and Tax in HTLV-1-transformed cells.
(A) Immunoblotting was performed with the indicated antibodies using cytoplasmic and nuclear fractions obtained from MT-2, HUT-102 and C8166 cells. (B) Immunofluorescence (IF) confocal microscopy was performed using MT-2 and C8166 cells with the indicated antibodies. The fluorescence intensity profile was plotted along the white arrow crossing the nucleus and represented in the graph showing overlap using NIS Element software. (C) PLA was performed using MT-2 and C8166 cells with Tax and UBE4B antibodies. (D) Graphical representation of the number of PLA signals with the secondary probe stained sample as a negative control. Unpaired Student’s t-test, **P <0.01.
Fig 3.
UBE4B enhances Tax-mediated activation of NF-κB.
(A-B) NF-κB and HTLV-1 LTR luciferase assays in 293T cells transfected with NF-κB-TATA or HTLV-1 luciferase, pRL-tk, Flag-UBE4B, FLAG-UBE4B P1140A and Flag-Tax as indicated. (C) NF-κB luciferase assays in 293T cells expressing either control shRNA or UBE4B shRNA were transfected with NF-κB-TATA luciferase, pRL-tk, Flag-Tax or stimulated with TNF for 8 h as indicated. (D-E) NF-κB and OCT-1 EMSA using nuclear extracts from MT-2 (D) and C8166 (E) cells expressing either control shRNA or UBE4B shRNA. C8166 cells were treated with Dox to induce UBE4B shRNA. UBE4B knockdown was confirmed by immunoblotting. Unpaired Student’s t-test, *P <0.05, **P <0.01, ns = not significant.
Fig 4.
UBE4B regulates IKK and canonical NF-κB signaling in HTLV-1-transformed cells.
(A-B) UBE4B was inducibly knocked down in C8166 and MT-2 cells by Dox treatment and immunoblotting was performed using lysates with the indicated antibodies. Canonical NF-κB and IKK activation was examined in panel A and noncanonical NF-κB activation was examined in panel B.
Fig 5.
Knockdown of UBE4B impairs Tax-induced NF-κB target gene expression.
(A-C) qRT-PCR of UBE4B, CD25, IRF-4, cIAP-2 and Tax mRNAs in MT-2, HUT-102, C8166 and TL-OM1 cells expressing control shRNA or UBE4B shRNA. (D) qRT-PCR of CD25, IRF-4, Tax and UBE4B mRNAs in Jurkat T cells expressing control shRNA or UBE4B shRNA and transduced with lentivirus expressing Tax. Unpaired Student’s t-test, *P <0.05, **P <0.01, ***P <0.001, ns = not significant.
Fig 6.
Tax activation of NF-κB is impaired in UBE4B KO cells.
(A-B) NF-κB and HTLV-1 LTR luciferase assays in wild-type (E2, H10) and UBE4B KO (F5, G12, H1) 293T cells transfected with NF-κB-TATA or HTLV-1 LTR luciferase, pRL-tk, and Tax as indicated. Immunoblotting was performed with lysates from transfected cells. (C) Immunoblotting was performed with the indicated antibodies using lysates from wild-type and UBE4B KO (clone H1) 293T cells transfected with Tax. One-way ANOVA with Dunnett’s post hoc test, ****P <0.0001.
Fig 7.
Knockdown of UBE4B promotes apoptotic cell death in HTLV-1-transformed cell lines.
(A) Immunoblotting was performed with the indicated antibodies using lysates from Jurkat, TL-OM1, MT-2, HUT-102 and C8166 cells expressing control shRNA or UBE4B shRNA. C8166 and TL-OM1 cells were treated with Dox to induce UBE4B shRNA. (B-F) Cell viability assay was performed at the indicated times with Jurkat, TL-OM1, MT-2, HUT-102 and C8166 cells expressing control shRNA or UBE4B shRNA. TL-OM1 cells were treated with Dox to induce UBE4B shRNA. Unpaired Student’s t-test, **P <0.01, ***P <0.001.
Fig 8.
UBE4B promotes the polyubiquitination of Tax.
(A) Ubiquitination assay was performed with purified 6xHis-Tax from lysates of 293T cells transfected with 6xHis-Tax and Flag-UBE4B as indicated. Immunoblotting was performed with the indicated antibodies. (B) Ubiquitination assay was performed with Tax immunoprecipitates from lysates of 293T cells transfected with Flag-Tax, Flag-UBE4B and Flag-UBE4B P1140A. (C) Ubiquitination assay was performed with Flag-Tax immunoprecipitates from lysates of 293T cells expressing control shRNA or UBE4B shRNA and transfected with Flag-Tax.
Fig 9.
Tax polyubiquitination is impaired in UBE4B KO cells.
(A-B) Ubiquitination assays were performed with Tax immunoprecipitates from lysates of wild-type and UBE4B KO (Clone H1) 293T cells transfected with Tax as indicated.
Fig 10.
UBE4B directly ubiquitinates Tax.
Schematic depicting the in vitro Tax ubiquitination assay protocol (left panel). Schematic was created with BioRender. In vitro ubiquitination assay was performed with the indicated recombinant proteins and immunoprecipitated Tax from lysates of transfected 293T cells. Following the Ub assay, Tax was immunoprecipitated from the reaction mixtures and immunoblotted with the indicated antibodies (right panel).
Fig 11.
Model of UBE4B regulation of Tax polyubiquitination and NF-κB activation.
Tax interacts with UBE4B, and UBE4B promotes Tax polyUb for downstream IKK and NF-κB activation. Created with BioRender.