Journal: Stem Cells International

Article Title: Electroacupuncture Improved Hippocampal Neurogenesis following Traumatic Brain Injury in Mice through Inhibition of TLR4 Signaling Pathway

doi: 10.1155/2017/5841814

Figure Lengend Snippet: EA treatment exerted an inhibitory effect on both of the two downstream pathways of TLR4 in the hippocampus posttrauma. Hippocampal TLR4, Myd88, TRAF6, TRAM, TRIF, and NF- κ B expression of the sham, TBI, TBI + EA, TBI + EA + LPS, TBI + EA + Veh, and sham + EA groups ( n = 3 in each group) were detected by WB at 35 days after TBI. (a–f) The six kinds of proteins in TLR4/Myd88 κ B and TLR4/TRIF/ κ B signaling pathways were upregualted in the TBI group compared with the sham group and the sham + EA group. EA treatment resulted in a significant decrease in the TBI + EA group, but the effects were attenuated by LPS administration in the TBI + EA + LPS group. No difference of the proteins between the TBI + EA and TBI + EA + Veh groups was observed. ∗ P < 0.05 versus the TBI group, † P < 0.05 versus the TBI + EA + LPS group.

Article Snippet: Membranes were blocked with 5% nonfat milk solution in tris-buffered saline with 0.1% Triton X-100 (TBST) for 1 hour and then incubated overnight at 4°C with appropriate primary antibodies as below: rabbit anti-mouse TLR4 antibody (1 : 1000, Thermo Fisher, PA5-23125, Rockford, IL, USA), rabbit anti-mouse myeloid differentiation factor 88 (Myd88) antibody (1 : 500, Santa Cruz, sc-17320, Dallas, TX, USA), rabbit anti-mouse TNFR-associated factor (TRAF6) antibody (1 : 1000, Novus Biological, NB100-56179, Littleton, CO, USA), rabbit anti-mouse toll/IL-1 receptor domain-containing adapter-induced interferon- β (TRIF) antibody (1 : 1000, Enzo Life Sciences, ALX-215-016, Farmingdale, NY, USA), rabbit anti-mouse TRIF-related adaptor molecule (TRAM) antibody (1 : 1000, OriGene, TA-306163, Rockville, MD, USA), rabbit anti-mouse nuclear factor- κ B (NF- κ B) p65 antibody (1 : 1000, GeneTex, GTX21893, Irvine, CA, USA), and rabbit anti- β -actin antibody (1 : 2000, Proteintech, 20536-1-AP, Rosemont, IL, USA).

Techniques: Expressing

Journal: Genes & Diseases

Article Title: Narciclasine induces colon carcinoma cell apoptosis by inhibiting the IL-17A/Act1/TRAF6/NF-κB signaling pathway

doi: 10.1016/j.gendis.2023.03.014

Figure Lengend Snippet: Ncs inhibits IL-17A/Act1/TRAF6/NF-κB signaling pathway in vitro . (A) CC HCT-116 cells were exposed to 0.5 μM Ncs for 24 h versus controls and a genome (KEGG) pathway enrichment was constructed by transcriptome sequencing. The sizes of the dots indicate negative log 10 ( P -value). Cells were exposed to Ncs for 24 h and the cell lysates were used to analyze the expression of IL-17 A, Act1, TRAF6, IκBɑ, IKKɑ/β, p-IκBɑ (Ser32), p-IKKɑ/β (Ser176/180), P65, and p-P65 (Ser536) in (B) HCT-116 cells and (C) SW-480 cells using western blotting. (D) HCT-116 and (E) SW-480 cells were exposed to 100 ng/mL Rh-IL-17 A or (F, G) si-IL-17 A in the absence and presence of Ncs (0.5 μM) for 24 h. Expression levels of IL-17 A, Act1, TRAF6, p-IκBɑ(Ser32), p-IKKɑ/β (Ser176/180), and p-P65 (Ser536) were detected by western blotting. Data are given as mean ± SD and are representative of 3 independent experiments. Statistical difference was analyzed using one-way ANOVA with a post hoc Bonferroni correction.

Article Snippet: Following the block, membranes were exposed to various primary antibodies, namely, IL-17 A (1:1,000, ab79056), NF-κB P65 (P65, 1:1,000, ab32536), Bax (1:1000, ab32503), Bcl-2 (1:1000, ab32124), Cytochrome c (1:1,000, ab13575), Caspase-3 (1:1000, ab32351), Caspase-8 (1:1,000, ab108333), Caspase-9 (1:1,000, ab202068), Ki-67 (1:1,000, ab92742), IκBα (1:1,000, ab32518), IKKα (1:1,000, ab178870), GAPDH (1:1,000, ab8245) (Abcam, USA), phospho-P65 (S536) (1:1,000, #3033), phospho-IKKα/β (S176/S180) (1:1000, #2697), phospho-IκBα (S32) (1:1,000, #2859), TRAF6 (1:1,000, #8028) (Cell Signaling Technology, USA), and Act1 (1:1,000, SC-100647) (Santa Cruz Biotechnology, USA).

Techniques: In Vitro, Construct, Sequencing, Expressing, Western Blot

Journal: Genes & Diseases

Article Title: Narciclasine induces colon carcinoma cell apoptosis by inhibiting the IL-17A/Act1/TRAF6/NF-κB signaling pathway

doi: 10.1016/j.gendis.2023.03.014

Figure Lengend Snippet: Molecular docking of IL-17 A and Act1, Act1 and TRAF6, as well as TRAF6 and p-IKKɑ/β (Ser176/180). (A) A three-dimensional diagram showing the binding conformation of IL-17 A (green) with Act1 (silvery white) and (B) of Act1 (silvery white) with TRAF6 (green). (C) Interactions between IL-17At and Act1, Act1 and TRAF6, TRAF6 and p-IKKɑ/β (Ser176/180) in the absence and presence of Ncs treatment were determined by co-immunoprecipitation assays.

Article Snippet: Following the block, membranes were exposed to various primary antibodies, namely, IL-17 A (1:1,000, ab79056), NF-κB P65 (P65, 1:1,000, ab32536), Bax (1:1000, ab32503), Bcl-2 (1:1000, ab32124), Cytochrome c (1:1,000, ab13575), Caspase-3 (1:1000, ab32351), Caspase-8 (1:1,000, ab108333), Caspase-9 (1:1,000, ab202068), Ki-67 (1:1,000, ab92742), IκBα (1:1,000, ab32518), IKKα (1:1,000, ab178870), GAPDH (1:1,000, ab8245) (Abcam, USA), phospho-P65 (S536) (1:1,000, #3033), phospho-IKKα/β (S176/S180) (1:1000, #2697), phospho-IκBα (S32) (1:1,000, #2859), TRAF6 (1:1,000, #8028) (Cell Signaling Technology, USA), and Act1 (1:1,000, SC-100647) (Santa Cruz Biotechnology, USA).

Techniques: Binding Assay, Immunoprecipitation

Journal: Genes & Diseases

Article Title: Narciclasine induces colon carcinoma cell apoptosis by inhibiting the IL-17A/Act1/TRAF6/NF-κB signaling pathway

doi: 10.1016/j.gendis.2023.03.014

Figure Lengend Snippet: IL-17 A, Act1, and TRAF6 expression were up-regulated in CC tissue and were significantly correlated with poor prognosis. (A) Representative immunohistochemistry staining of IL-17 A, Act1, TRAF6 expression in CC tissue and non-cancer tissues. Scale bar = 50 μm. (B) Statistical analysis of immunohistochemical scores of IL-17 A, Act1, and TRAF6 in CC tissue and non-cancer tissues. (C) Percentage of high expression and low expression of IL-17 A, Act1, and TRAF6 in CC tissue and non-cancer tissues. (D) Pearson correlative analysis of staining scores for IL-17 A and Act1, L-17 A and TRAF6, as well as Act1 and TRAF6 in the CC tissue. (E) Overall survival durations of CC patients after Kaplan–Meier analysis were defined as low or high IL-17 A, Act1, or TRAF6 expression groups. Data are given as mean ± SD.

Article Snippet: Following the block, membranes were exposed to various primary antibodies, namely, IL-17 A (1:1,000, ab79056), NF-κB P65 (P65, 1:1,000, ab32536), Bax (1:1000, ab32503), Bcl-2 (1:1000, ab32124), Cytochrome c (1:1,000, ab13575), Caspase-3 (1:1000, ab32351), Caspase-8 (1:1,000, ab108333), Caspase-9 (1:1,000, ab202068), Ki-67 (1:1,000, ab92742), IκBα (1:1,000, ab32518), IKKα (1:1,000, ab178870), GAPDH (1:1,000, ab8245) (Abcam, USA), phospho-P65 (S536) (1:1,000, #3033), phospho-IKKα/β (S176/S180) (1:1000, #2697), phospho-IκBα (S32) (1:1,000, #2859), TRAF6 (1:1,000, #8028) (Cell Signaling Technology, USA), and Act1 (1:1,000, SC-100647) (Santa Cruz Biotechnology, USA).

Techniques: Expressing, Immunohistochemistry, Staining, Immunohistochemical staining

Journal: Genes & Diseases

Article Title: Narciclasine induces colon carcinoma cell apoptosis by inhibiting the IL-17A/Act1/TRAF6/NF-κB signaling pathway

doi: 10.1016/j.gendis.2023.03.014

Figure Lengend Snippet: Correlation analyses of IL-17 A, Act1 and TRAF6 expression in colorectal cancer with clinicopathologic features.

Article Snippet: Following the block, membranes were exposed to various primary antibodies, namely, IL-17 A (1:1,000, ab79056), NF-κB P65 (P65, 1:1,000, ab32536), Bax (1:1000, ab32503), Bcl-2 (1:1000, ab32124), Cytochrome c (1:1,000, ab13575), Caspase-3 (1:1000, ab32351), Caspase-8 (1:1,000, ab108333), Caspase-9 (1:1,000, ab202068), Ki-67 (1:1,000, ab92742), IκBα (1:1,000, ab32518), IKKα (1:1,000, ab178870), GAPDH (1:1,000, ab8245) (Abcam, USA), phospho-P65 (S536) (1:1,000, #3033), phospho-IKKα/β (S176/S180) (1:1000, #2697), phospho-IκBα (S32) (1:1,000, #2859), TRAF6 (1:1,000, #8028) (Cell Signaling Technology, USA), and Act1 (1:1,000, SC-100647) (Santa Cruz Biotechnology, USA).

Techniques: Expressing

Journal: Genes & Diseases

Article Title: Narciclasine induces colon carcinoma cell apoptosis by inhibiting the IL-17A/Act1/TRAF6/NF-κB signaling pathway

doi: 10.1016/j.gendis.2023.03.014

Figure Lengend Snippet: Correlation of the expression of IL-17 A, Act1, and TRAF6 in CC with clinicopathologic features. The correlation between IL-17 A, Act1, and TRAF6 expression and CC patients' clinicopathologic characteristics in IHC analysis. There were obvious relationships between IL-17 A, TRAF6 expression and differentiation, the expression of Act1 and lymph node metastases. Statistical significance was determined using the χ 2 -test.

Article Snippet: Following the block, membranes were exposed to various primary antibodies, namely, IL-17 A (1:1,000, ab79056), NF-κB P65 (P65, 1:1,000, ab32536), Bax (1:1000, ab32503), Bcl-2 (1:1000, ab32124), Cytochrome c (1:1,000, ab13575), Caspase-3 (1:1000, ab32351), Caspase-8 (1:1,000, ab108333), Caspase-9 (1:1,000, ab202068), Ki-67 (1:1,000, ab92742), IκBα (1:1,000, ab32518), IKKα (1:1,000, ab178870), GAPDH (1:1,000, ab8245) (Abcam, USA), phospho-P65 (S536) (1:1,000, #3033), phospho-IKKα/β (S176/S180) (1:1000, #2697), phospho-IκBα (S32) (1:1,000, #2859), TRAF6 (1:1,000, #8028) (Cell Signaling Technology, USA), and Act1 (1:1,000, SC-100647) (Santa Cruz Biotechnology, USA).

Techniques: Expressing

Journal: PLoS Pathogens

Article Title: GPATCH3 negatively regulates RLR-mediated innate antiviral responses by disrupting the assembly of VISA signalosome

doi: 10.1371/journal.ppat.1006328

Figure Lengend Snippet: (A) 293T cells (2 x 10 6 ) were cotransfected with Flag-VISA (3 μg) and HA-tagged TBK1, TRAF6 or TRAF3 (3 μg), together with increasing amounts of GPATCH3 (0, 0.5 μg, 1.5μg). Coimmunoprecipitation and immunoblotting were performed with the indicated antibodies. (B) Wild-type or GPACH3-deficient cells (1.5 x 10 7 ) were left uninfected or infected with SeV for the indicated times. Coimmunoprecipitation and immunoblotting were performed with the indicated antibodies.

Article Snippet: HEK293T (Transformed Human Embryonic Kidney 293 cell line, ATCC) cells, A549 (Human Lung Adenocarcinoma cell line, ATCC) cells, PBMCs (Peripheral Blood Mononuclear Cells, Allcells), Human recombinant TNFα (R&D Systems), poly(I:C) (InvivoGen), Lipofectamine 2000 (Invitrogen), dual-specific luciferase assay kits (Promega), mouse monoclonal antibodies against Flag and β-actin (Sigma), HA (Origene), AIF, LMNB1 and TBK1 (Abcam), IRF-3 (Proteintech), VISA (Bethyl Laboratories), TRAF6 and GPATCH3 (Y-20) (Santa Cruz Biotechnology), phospho-IRF3 (Ser396), phospho-TBK1 (Ser172) and phospho-IKKα (Ser176)/β (Ser177) (Cell Signaling Technology) were purchased from the indicated companies.

Techniques: Western Blot, Infection

Journal: eLife

Article Title: PIMT is a novel and potent suppressor of endothelial activation

doi: 10.7554/elife.85754

Figure Lengend Snippet: Figure 3. PIMT functionally interacts with TRAF6. (A) EA.hy926 cells were transfected with NF-κB luciferase reporter together with indicated plasmids. Cells were harvested 48 hr later to determine luciferase activity (n=4), ***p<0.001, and two-way ANOVA coupled with Tukey’s post hoc test. Expression of transfected proteins was detected by western blot. (B) HEK-293T cells were transfected with a combination of indicated plasmids. 48 hr after transfection, immunoprecipitation was performed using anti-Flag antibody, followed by western blot to detect protein interaction. (C) HEK-293T cells were transfected with Flag-TRAF6 and Myc-PIMT expression vectors. 48 hr after transfection, immunoprecipitation was performed using anti-Flag antibody, followed by western blot to detect the interaction of PIMT with TRAF6. (D) HEK-293T cells were transfected with HA-TRAF6 and Flag-PIMT vectors. 48 hr after transfection, immunoprecipitation was performed using anti-HA antibody, followed by western blot to detect protein interaction. (E) Human umbilical vein endothelial cells (HUVECs) were transduced with lentivirus expressing the control shRNA (sh-Ctrl) or TRAF6 shRNA (sh-TRAF6) for 72 hr, followed by stimulation with either vehicle or LPS (1 μg/ml) for 30 min. Cell lysates were then collected for immunoprecipitation using anti-PIMT

Article Snippet: AntiGAPDH (10494–1- AP), anti- alpha Tubulin (11224–1- AP), and anti- TRAF6 (66498–1- Ig) antibodies were acquired from Proteintech Group.

Techniques: Transfection, Luciferase, Activity Assay, Expressing, Western Blot, Immunoprecipitation, Transduction, Control, shRNA

Journal: eLife

Article Title: PIMT is a novel and potent suppressor of endothelial activation

doi: 10.7554/elife.85754

Figure Lengend Snippet: Figure 4. PIMT suppresses TRAF6 function through methylation of TRAF6 at N350. (A) HEK-293T cells were transfected with TRAF6 and PIMT constructs. Immunoprecipitation was performed 48 hr after transfection using anti-Flag antibody, followed by western blot using anti-HA antibody to detect TRAF6 oligomerization. (B) HEK- 293T cells were transfected with HA-ubiquitin (HA-Ub) and Flag-TRAF6 in the presence of Myc-tagged wild-type

Article Snippet: AntiGAPDH (10494–1- AP), anti- alpha Tubulin (11224–1- AP), and anti- TRAF6 (66498–1- Ig) antibodies were acquired from Proteintech Group.

Techniques: Methylation, Transfection, Construct, Immunoprecipitation, Western Blot, Ubiquitin Proteomics

Journal: Cell Death & Disease

Article Title: Cereblon negatively regulates TLR4 signaling through the attenuation of ubiquitination of TRAF6

doi: 10.1038/cddis.2016.226

Figure Lengend Snippet: CRBN interacts with TRAF6 and inhibits the ubiquitination of TRAF6. ( a ) HEK293T cells were transfected with mock, HA-CRBN, or Flag-TRAF6, as indicated. At 38 h after transfection, transfected cells were extracted, immunoprecipitated with anti-Flag antibody, and then immunoblotting was performed with anti-Flag or anti-HA antibodies. ( b ) A schematic presentation of TRAF6 wt and its truncated mutants used in this study: TRAF6 110-522, TRAF6 260-522, and TRAF6 349-522. ( c ) HEK293T cells were transfected with HA-CRBN, Flag-TRAF6 wt, Flag-TRAF6 110-522, Flag-TRAF6 260-522, or Flag-TRAF6 349-522, as indicated. At 38 h after transfection, transfected cells were extracted, immunoprecipitated with an anti-Flag antibody, and then immunoblotting was performed with anti-Flag or anti-HA antibodies. ( d ) HEK293T cells were transfected with mock, Myc-CRBN, Flag-TRAF6, or HA-Ub, as indicated. At 38 h after transfection, transfected cells were extracted, immunoprecipitated with an anti-Flag antibody, and then immunoblotting was performed with anti-Flag, anti-Myc, or anti-HA antibodies. ( e ) HEK293T cells were transfected with mock, HA-Ub, and Flag-TRAF6 in the absence or presence of different concentrations of Myc-CRBN. At 38 h after transfection, transfected cells were extracted, immunoprecipitated with an anti-Flag antibody, and then immunoblotting was performed with anti-Flag, anti-Myc, or anti-HA antibodies. ( f ) THP-1 cells were infected with a lentivirus containing shRNA targeted to human TRAF6 or control shRNA sequences. Two weeks post-infection, endogenous expression of TRAF6 protein was analyzed in TRAF6 KD and control (Ctrl) THP-1 cells. ( g ) TRAF6 KD and Ctrl THP-1 cells were transfected with mock, Flag-TRAF6, or HA-CRBN vectors, as indicted, together with pBIIx-luc and Renilla luciferase. Twenty-four hours after transfection, cells were untreated or treated with LPS (200 ng/ml) for 6 h and then analyzed for luciferase activity. Results are expressed as the fold induction in luciferase activity relative to that in untreated cells. All error bars represent S.D. of the mean from triplicate samples. * P <0.05. ( h ) TRAF6 KD and Ctrl THP-1 cells were transfected with mock, Flag-TRAF6, or HA-CRBN vectors, as indicated, and treated with or without LPS (200 ng/ml) for 9 h, then, production of IL-6 was analyzed by ELISA. All error bars represent S.D. of the mean from triplicate samples. * P <0.05 and ** P <0.01

Article Snippet: Lentivirus containing small hairpin RNA (shRNA) targeting human CRBN (sc-78528-V), lentivirus containing shRNA targeting human TRAF6 (sc-36717-V), or control shRNA lentivirus (sc-108080) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Transfection, Immunoprecipitation, Western Blot, Infection, shRNA, Expressing, Luciferase, Activity Assay, Enzyme-linked Immunosorbent Assay

Journal: Cell Death & Disease

Article Title: Cereblon negatively regulates TLR4 signaling through the attenuation of ubiquitination of TRAF6

doi: 10.1038/cddis.2016.226

Figure Lengend Snippet: CRBN inhibits ubiquitination of TAB2. ( a ) HEK293T cells were transfected with mock, Myc-CRBN, Flag-TAB2, or HA-Ub, as indicated. At 38 h after transfection, transfected cells were extracted, immunoprecipitated with anti-Flag antibody, and then immunoblotting was performed with anti-Flag, anti-Myc, or anti-HA antibodies. ( b ) HEK293T cells were transfected with mock, HA-Ub, and Flag-TAB2 in the absence or presence of different concentrations of Myc-CRBN. At 38 h after transfection, transfected cells were extracted, immunoprecipitated with an anti-Flag antibody, and then immunoblotting was performed with anti-Flag, anti-Myc, or anti-HA antibodies. ( c ) CRBN +/+ or CRBN −/− MEF cells were stimulated without or with LPS for different periods of time, as indicated, then subjected to extraction, and western blotting was performed with the antibodies indicated on the left. The band intensity of p-IKK α / β was analyzed with Image J (bottom). All error bars represent S.D. of the mean from three independent experiments * P <0.05 versus without LPS. ( d ) TRAF6 KD and Ctrl THP-1 cells were transfected with mock, Flag-TRAF6 wt, Flag-TAB2, or HA-CRBN vectors, as indicted, together with pBIIx-luc and Renilla luciferase. Twenty-four hours after transfection, cells were untreated or treated with LPS (200 ng/ml) for 6 h and then analyzed for luciferase activity. Results are expressed as the fold induction in luciferase activity relative to that in untreated cells. All error bars represent S.D. of the mean from triplicate samples. * P <0.05. ( e ) TRAF6 KD and Ctrl THP-1 cells were transfected with mock, Flag-TRAF6 wt, Flag-TAB2, or HA-CRBN vectors, as indicted, and treated with or without LPS (200 ng/ml) for 9 h, and production of IL-6 was analyzed by ELISA. All error bars represent S.D. of the mean from triplicate samples. * P <0.05 and ** P <0.01

Article Snippet: Lentivirus containing small hairpin RNA (shRNA) targeting human CRBN (sc-78528-V), lentivirus containing shRNA targeting human TRAF6 (sc-36717-V), or control shRNA lentivirus (sc-108080) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Transfection, Immunoprecipitation, Western Blot, Luciferase, Activity Assay, Enzyme-linked Immunosorbent Assay

Journal: Cell Death & Disease

Article Title: Cereblon negatively regulates TLR4 signaling through the attenuation of ubiquitination of TRAF6

doi: 10.1038/cddis.2016.226

Figure Lengend Snippet: Crbn −/− mice exhibit a higher mortality rate following LPS challenge. ( a ) Crbn −/− ( n =8) and Crbn +/+ ( n =8) mice were injected intraperitoneally with 7.5 mg/kg LPS in 100 μ l of PBS. Survival was monitored for 7 days after LPS challenge. ( b,c ) Serum from mice taken at different times after LPS challenge was isolated, and the concentrations of the TNF- α ( b ) and IL-6 ( c ) cytokines were measured with a BD Cytometric Bead Array. Error bars represent the mean±S.D. of eight samples. * P <0.05 ( d ) A schematic model of the negative regulation of CRBN in TLR4 signaling. Upon TLR4 stimulation, ubiquitinated TRAF6 is associated with the TAB2–TAK–TAB1 complex though the interaction between its poly-ubiquitinated chain and TAB2, TAB2 is ubiquitinated, and the complex facilities the activation of TAK1. Simultaneously, the IKK complex is associated with the former complex through the poly-ubiquitinated chain and TAB2, leading to the activation of IKKs (left). In contrast, the interaction between CRBN and TRAF6 may lead to the attenuation of ubiquitination of TRAF6 by masking the K124 residue of TRAF6 and inhibiting the association of the TAK1–TAB1–TAB2 complex to TRAF6, leading to inhibition of ubiquitination of TAB2 and decreased association of the IKK complex with ubiquitinated TAB2 (right)

Article Snippet: Lentivirus containing small hairpin RNA (shRNA) targeting human CRBN (sc-78528-V), lentivirus containing shRNA targeting human TRAF6 (sc-36717-V), or control shRNA lentivirus (sc-108080) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Injection, Isolation, Activation Assay, Inhibition

Journal: Cellular Oncology (Dordrecht, Netherlands)

Article Title: Decitabine promotes degradation of DNMT1 and EZH2 via the ubiquitination pathway and inhibits colorectal cancer progression

doi: 10.1007/s13402-025-01136-8

Figure Lengend Snippet: TRAF6 mediates DAC-induced DNMT1 ubiquitination and degradation. (A) Reduced TRAF6 mRNA expression in colorectal cancer tissues compared to adjacent normal tissues (TCGA dataset; RNA-seq analysis; **** p < 0.0001). (B) Kaplan-Meier analysis reveals prolonged post-progression survival (PPS) in patients with high TRAF6 expression versus low expression (log-rank test, p = 0.0061). (C) Inverse correlation between DNMT1 and TRAF6 mRNA expression in TCGA colorectal cohorts (Spearman’s r = -0.1603, p = 0.0008061). (D) Immunofluorescence showing co-localization of DNMT1 (green) and TRAF6 (red) in HCT8 and SW620 (DAPI: blue; scale bar:100 μm). (E) TRAF6 protein upregulation in DAC-treated cells. (F) TRAF6 knockdown (siTRAF6) significantly elevated DNMT1 protein levels. (G) IP-Western blot showing reduced DNMT1 ubiquitination upon TRAF6 knockdown (anti-Ub antibody; IgG as IP negative control). (H) TRAF6 knockdown blocks DAC-induced DNMT1 degradation I , J. TRAF6 protein levels inversely correlate with DNMT1 modulation. K-M. qRT-PCR reveals TRAF6 transcriptional upregulation following DNMT1 knockdown (siDNMT1) or DAC treatment (500nM, 48 h), and suppression by DNMT1 OE(* p < 0.05,** p < 0.01, *** p < 0.001, **** p < 0.0001). N. DNMT1 induces hypermethylation of the TRAF6 promoter. Methylation-specific PCR (MSP) using methylated (M) and unmethylated (U) primers. DNMT1 OE enhances methylation (intensified M-band). O. Dual-luciferase assays show DNMT1 knockdown or treated with DAC increases TRAF6 promotor activity in HEK293T cells(** p < 0.01)

Article Snippet: Cells were washed three in PBS and blocked for 1 h with 5% BSA in PBS and then incubated with primary antibodies DNMT1 (abcam, ab188453) and TRAF6 (santa cruz, sc-8409) at 4 °C overnight.

Techniques: Ubiquitin Proteomics, Expressing, RNA Sequencing, Immunofluorescence, Knockdown, Western Blot, Negative Control, Quantitative RT-PCR, Methylation, Luciferase, Activity Assay

Journal: Cellular Oncology (Dordrecht, Netherlands)

Article Title: Decitabine promotes degradation of DNMT1 and EZH2 via the ubiquitination pathway and inhibits colorectal cancer progression

doi: 10.1007/s13402-025-01136-8

Figure Lengend Snippet: DNMT1 silences TRAF6 to block EZH2 ubiquitination. A-C. DNMT1 modulation regulates EZH2 protein levels in CRC cells: Western blot analysis of EZH2 in DNMT1-overexpressing and DNMT1-knockdown( A , B ), DAC-treated ( C ) cells. D-G. qRT-PCR analysis of EZH2 mRNA in HCT8 ( D ) and SW480 ( E ) cells with DNMT1 modulation (ns: not significant). H-I. Cycloheximide (CHX, 50 µg/mL) chase assay showing reduced EZH2 half-life in DNMT1-knockdown HCT8 cells. J. Proteasome inhibitor MG132 (10 µM, 6 h) rescues EZH2 protein levels in DNMT1-depleted cells. K-L. Endogenous ubiquitination assays showing increased polyubiquitinated EZH2 in DNMT1-knockdown or treated with DAC cells (IP with anti-EZH2 followed by anti-Ub Western blot; input: 5% lysate). M. TCGA data analysis reveals significantly elevated EZH2 mRNA expression in colorectal cancer (CRC) tissues compared to adjacent normal tissues. N. Expression correlation analysis demonstrates a significant inverse relationship between TRAF6 and EZH2 mRNA levels in colorectal cohorts ( p < 0.0001). O , P. Overexpression of TRAF6 enhances polyubiquitination of EZH2, as detected by ubiquitin (Ub) immunoblotting in CRC cells. TRAF6 knockdown reduces EZH2 ubiquitination levels. Lysates were immunoprecipitated (IP) with anti-EZH2 and immunoblotted (IB) with anti-Ub. Q. Co-IP assay demonstrating strengthened EZH2-TRAF6 interaction in DNMT1-depleted cells (lysates immunoprecipitated with anti-EZH2, blotted for TRAF6; input: 5% lysate). R. Dual inhibition of TRAF6 and DNMT1 rescues EZH2 hyperubiquitination (IP-Western blot with siTRAF6/siDNMT1 co-treatment

Article Snippet: Cells were washed three in PBS and blocked for 1 h with 5% BSA in PBS and then incubated with primary antibodies DNMT1 (abcam, ab188453) and TRAF6 (santa cruz, sc-8409) at 4 °C overnight.

Techniques: Blocking Assay, Ubiquitin Proteomics, Western Blot, Knockdown, Quantitative RT-PCR, Expressing, Over Expression, Immunoprecipitation, Co-Immunoprecipitation Assay, Inhibition

Journal: Cellular Oncology (Dordrecht, Netherlands)

Article Title: Decitabine promotes degradation of DNMT1 and EZH2 via the ubiquitination pathway and inhibits colorectal cancer progression

doi: 10.1007/s13402-025-01136-8

Figure Lengend Snippet: EZH2 is a downstream effector of DNMT1 in CRC progression. (A) Western blot analysis showing that EZH2 knockdown reverses DNMT1-induced upregulation of cyclinD1 and cyclinE2 in HCT8 and SW480 cells. (B) Colony formation assays demonstrating that EZH2 depletion abolishes DNMT1-driven proliferation in HCT8 cells (** p < 0.01 vs. DNMT1-OE + siControl). C-D. Colony formation assays demonstrating that TRAF6 overexpression abolishes DNMT1/EZH2-driven proliferation in HCT8 cells (** p < 0.01 vs. DNMT1-OE + Vector, ** p < 0.01 vs. EZH2-OE + Vector). E. Transwell migration assays showing suppression of DNMT1-enhanced motility by EZH2 knockdown in HCT8 cells(*** p < 0.001 vs. DNMT1-OE + siControl); Matrigel invasion assays confirming EZH2-dependent regulation of DNMT1-induced invasiveness in SW480 cells(** p < 0.01 vs. DNMT1-OE + siControl; scale bar: 100 μm). F. Transwell migration assays showing suppression of DNMT1-enhanced motility by TRFA6 overexpression in HCT8 cells(** p < 0.01 vs. DNMT1-OE + Vector); Matrigel invasion assays confirming TRAF6-dependent regulation of DNMT1-induced invasiveness in HCT8 cells(*** p < 0.001 vs. DNMT1-OE + Vector; scale bar: 100 μm). G. Transwell migration assays showing suppression of EZH2-enhanced motility by TRFA6 overexpression in HCT8 cells(*** p < 0.001 vs. EZH2-OE + Vector); Matrigel invasion assays confirming TRAF6-dependent regulation of EZH2-induced invasiveness in HCT8 cells(*** p < 0.001 vs. EZH2-OE + Vector; scale bar: 100 μm)

Article Snippet: Cells were washed three in PBS and blocked for 1 h with 5% BSA in PBS and then incubated with primary antibodies DNMT1 (abcam, ab188453) and TRAF6 (santa cruz, sc-8409) at 4 °C overnight.

Techniques: Western Blot, Knockdown, Over Expression, Plasmid Preparation, Migration

Journal: Cellular Oncology (Dordrecht, Netherlands)

Article Title: Decitabine promotes degradation of DNMT1 and EZH2 via the ubiquitination pathway and inhibits colorectal cancer progression

doi: 10.1007/s13402-025-01136-8

Figure Lengend Snippet: Effects of Decitabine (DAC) Alone and in Combination with Tazemetostat (TAZ) on the Biological Behaviors of SW480 Cells and Analysis of Related Molecular Expressions. (A) Representative images of colonies formed by SW480 cells treated with DMSO (vehicle control), decitabine (DAC, 500 nM) alone, tazemetostat (TAZ, 1 µM) alone, or the combination (DAC + TAZ) (*** p < 0.001). (B) Representative images (left) and quantitative analysis (right) of the migration (upper panel) and invasion (lower panel) of SW480 cells after treatment with DMSO (vehicle control), decitabine (DAC, 500 nM) alone, tazemetostat (TAZ, 1 µM) alone, or the combination (DAC + TAZ) (** p < 0.01, *** p < 0.001). (C) The expression levels of DNMT1, EZH2 and TRAF6 in colorectal cancer tissues and adjacent non-tumor tissues. (D) Regulatory Relationships among Decitabine, TRAF6, DNMT1 and EZH2

Article Snippet: Cells were washed three in PBS and blocked for 1 h with 5% BSA in PBS and then incubated with primary antibodies DNMT1 (abcam, ab188453) and TRAF6 (santa cruz, sc-8409) at 4 °C overnight.

Techniques: Control, Migration, Expressing