Journal: Nucleic Acids Research

Article Title: The neurodegenerative diseases ALS and SMA are linked at the molecular level via the ASC-1 complex

doi: 10.1093/nar/gky1093

Figure Lengend Snippet: ALS- and SMA-causative proteins in the RNAP II/U1 snRNP machinery

Article Snippet: Polyclonal antibodies against EWSR1 (cat # A300-418A), MATR3 (cat # A300-591A), HA (cat # A190-108A), ASCC1 (cat # A303-871A), ASCC2 (cat # A304-020A), ASCC3 (cat # A304-014A) and TRIP4 (cat # A300-203A) were from Bethyl (Montgomery, TX, USA), and the polyclonal antibody against TAF15 was from Novus (Littleton, CO) (cat # NB100-567).

Techniques:

Journal: Nucleic Acids Research

Article Title: The neurodegenerative diseases ALS and SMA are linked at the molecular level via the ASC-1 complex

doi: 10.1093/nar/gky1093

Figure Lengend Snippet: Disease-causing mutations in FUS or TRIP4 disrupt interactions of ASC-1 complex components with the RNAP II/U1 snRNP machinery. ( A ) Purified recombinant GST-FUS or GST-DDX39B was added to FUS KO extract, and RNAP II was IP’d followed by Westerns with antibodies to the ASC-1 components. The asterisk indicates degradation products of GST-FUS. Endogenous FUS, GST-FUS, and GST-DDX39B were detected by the FUS antibody, as this antibody recognizes GST-DDX39B due to the GST tag common to both proteins. ( B ) Three independent replicates of the data shown in (A) were quantitated. The colored bars in the graph show the mean values of fold change for the indicated proteins. Error bars represent standard deviations. * P < 0.05, n.s., not significant (two-tailed Student's t -test). ( C and D ), same as (A and B), except that GST-FUS G156E or FUSR 514G was used for add-backs. ( E ) HA-DDX39B, HA-TRIP4 or HA-TRIP4 1-254 was expressed in HeLa cells followed by IP/westerns with the indicated antibodies. ( F ) Quantitation of three independent replicates of data shown in (E). ** P < 0.01 (two-tailed Student's t -test.)

Article Snippet: Polyclonal antibodies against EWSR1 (cat # A300-418A), MATR3 (cat # A300-591A), HA (cat # A190-108A), ASCC1 (cat # A303-871A), ASCC2 (cat # A304-020A), ASCC3 (cat # A304-014A) and TRIP4 (cat # A300-203A) were from Bethyl (Montgomery, TX, USA), and the polyclonal antibody against TAF15 was from Novus (Littleton, CO) (cat # NB100-567).

Techniques: Purification, Recombinant, Two Tailed Test, Quantitation Assay

Journal: Biomarker Research

Article Title: Histone modification-regulated LncRNA DLEU1 interacts with ASCC2/ALKBH3 complex to drive DNA repair, antioxidant homeostasis and glucose metabolism in gastric cancer

doi: 10.1186/s40364-025-00867-y

Figure Lengend Snippet: DLEU1 facilitates ASCC2 nuclear translocation and promotes ASCC2–ALKBH3 interaction. A . Silver staining (left panel) analysis of proteins isolated from AGS cells using ChIRP with DLEU1 or the control LacZ probes. The red rectangle highlighted the potential presence of ASCC2 in the DLEU1 complex, subsequently confirmed by LC/MS mass spectrometry analysis (right panel). B - C . RNA pull-down ( B ) and RIP assays ( C ) demonstrated the interaction between ASCC2 and DLEU1 in AGS and HGC27 cells. D . Immunofluorescence staining showed co-localization of DLEU1 with ASCC2, Scale bar = 10 μm. E . Western blot results showed that overexpression of DLEU1 increased ASCC2 protein levels in MGC803 and AGS cells, whereas knockdown of DLEU1 decreased ASCC2 protein levels in HGC27 and MKN45 cells. F - G . Nuclear-cytoplasmic fractions ( F ) and Immunofluorescence staining ( G ) demonstrate increased nuclear localization of ASCC2 in DLEU1 overexpressed cells. Scale bar = 10 μm. H . Endogenous co-immunoprecipitation assays reveal enhanced ASCC2–ALKBH3 binding following DLEU1 overexpression. I . Western blot analysis showed changes in ASCC2 and ALKBH3 protein expression in AGS with DLEU1 overexpression or combined DLEU1 overexpression with ASCC2 knockdown(left panel), and in HGC27 cells with DLEU1 knockdown or combined DLEU1 knockdown with ASCC2 overexpression (right panel). *, P < 0.05; **, P < 0.01; ***, P < 0.001; ns, not significant. See also Figures

Article Snippet: The staining sequence and dye combination are as follows: DLEU1 probe ( NR_109973.1 , 5-1627, working solution, Alpha X Bio, China) - TSA520; E2F1 (1/200 dilution, Proteintech, Cat. #66515-1-Ig, RRID: AB_2881878) - TSA480; ALKBH3 (1/200 dilution, Proteintech, Cat. #12292-1-AP, RRID: AB_11125161) - TSA620; and ASCC2 (1/200 dilution, Proteintech, Cat.#11529-1-AP, RRID: AB_2059358) - TSA780.

Techniques: Translocation Assay, Silver Staining, Isolation, Control, Liquid Chromatography with Mass Spectroscopy, Mass Spectrometry, Immunofluorescence, Staining, Western Blot, Over Expression, Knockdown, Immunoprecipitation, Binding Assay, Expressing

Journal: Biomarker Research

Article Title: Histone modification-regulated LncRNA DLEU1 interacts with ASCC2/ALKBH3 complex to drive DNA repair, antioxidant homeostasis and glucose metabolism in gastric cancer

doi: 10.1186/s40364-025-00867-y

Figure Lengend Snippet: DLEU1 promotes G6PD transcription via ASCC2/ALKBH3-mediated recruitment of E2F1. A - B . qRT-PCR and Western blot analysis of E2F1 expression in AGS and MGC803 cells upon ALKBH3 overexpression. C . Time-course analysis of E2F1 mRNA levels in AGS and MGC803 cells transfected with vector or ALKBH3. D - E . RT-qPCR and western blot analyzed of E2F1 and G6PD expression in AGS and MGC803 cells following DLEU1 overexpression or DLEU1 overexpression combined with ASCC2 knockdown. F - G . RT-qPCR and western blot analyzed of G6PD levels in AGS and MGC803 cells with E2F1 overexpression. H . qRT-PCR analysis of G6PD mRNA expression in AGS and MGC803 cells after ALKBH3 overexpression or co-transfection with ALKBH3 and shRNA targeting E2F1 (shE2F1). I . Western blot analysis of ASCC2, G6PD and E2F1 expression in AGS and MGC803 cells with ALKBH3 overexpression or ALKBH3 overexpression combined with E2F1 knockdown. J . ChIP-qPCR analysis confirmed E2F1 binding to the G6PD promoter and intron 2 regions in AGS cells. K . Luciferase reporter assay showed the transcriptional activity of wild-type and mutant G6PD promoter and intron 2 regions in AGS cells. L . Luciferase assays demonstrate that knockdown of DLEU1 , ASCC2, or ALKBH3 attenuates E2F1-mediated activation of G6PD transcription. L - M . ChIP-qPCR and luciferase reporter validated E2F1 binding to an intronic region of ASCC2 in AGS cells. M . Luciferase assays indicate that knockdown of DLEU1 , ASCC2, or ALKBH3 reduces E2F1-driven ASCC2 transcription. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ns, not significant

Article Snippet: The staining sequence and dye combination are as follows: DLEU1 probe ( NR_109973.1 , 5-1627, working solution, Alpha X Bio, China) - TSA520; E2F1 (1/200 dilution, Proteintech, Cat. #66515-1-Ig, RRID: AB_2881878) - TSA480; ALKBH3 (1/200 dilution, Proteintech, Cat. #12292-1-AP, RRID: AB_11125161) - TSA620; and ASCC2 (1/200 dilution, Proteintech, Cat.#11529-1-AP, RRID: AB_2059358) - TSA780.

Techniques: Quantitative RT-PCR, Western Blot, Expressing, Over Expression, Transfection, Plasmid Preparation, Knockdown, Cotransfection, shRNA, ChIP-qPCR, Binding Assay, Luciferase, Reporter Assay, Activity Assay, Mutagenesis, Activation Assay

Journal: Biomarker Research

Article Title: Histone modification-regulated LncRNA DLEU1 interacts with ASCC2/ALKBH3 complex to drive DNA repair, antioxidant homeostasis and glucose metabolism in gastric cancer

doi: 10.1186/s40364-025-00867-y

Figure Lengend Snippet: Inhibition of ASCC2 abrogates DLEU1 -mediated DNA damage repair and apoptosis resistance in GC cells. A . Proliferation assays in AGS and MGC803 with DLEU1 overexpression, with or without ASCC2 knockdown, and in HGC27 and MKN45 cells with DLEU1 knockdown, with or without ASCC2 overexpression. B - C . Comet assay, Western blot analysis of γ-H2AX protein with DLEU1 overexpression or DLEU1 overexpression combined with ASCC2 knockdown. Scale bar = 50 μm. D - E . Immunofluorescence staining of γH2AX in AGS cells treated with Etoposide. Representative images at 0, 4, 8, and 12 h after treatment are shown. Quantification of average foci number is shown in figure E . Scale bar = 100 μm. F . Apoptosis analysis by flow cytometry in AGS and MGC803 with DLEU1 overexpression or DLEU1 overexpression combined with ASCC2 knockdown. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ns, not significant

Article Snippet: The staining sequence and dye combination are as follows: DLEU1 probe ( NR_109973.1 , 5-1627, working solution, Alpha X Bio, China) - TSA520; E2F1 (1/200 dilution, Proteintech, Cat. #66515-1-Ig, RRID: AB_2881878) - TSA480; ALKBH3 (1/200 dilution, Proteintech, Cat. #12292-1-AP, RRID: AB_11125161) - TSA620; and ASCC2 (1/200 dilution, Proteintech, Cat.#11529-1-AP, RRID: AB_2059358) - TSA780.

Techniques: Inhibition, Over Expression, Knockdown, Single Cell Gel Electrophoresis, Western Blot, Immunofluorescence, Staining, Flow Cytometry

Journal: Biomarker Research

Article Title: Histone modification-regulated LncRNA DLEU1 interacts with ASCC2/ALKBH3 complex to drive DNA repair, antioxidant homeostasis and glucose metabolism in gastric cancer

doi: 10.1186/s40364-025-00867-y

Figure Lengend Snippet: DLEU1 knockdown suppresses tumor growth by targeting ASCC2 and G6PD in xenograft models. A . Schematic representation of the xenograft experiment. MKN45 cells with stable knockdown of DLEU1 ( shDLEU1 ) or co-knockdown of DLEU1 and ASCC2 ( shDLEU1 + shASCC2 ) were subcutaneously implanted into nude mice. G6PD inhibitor (G6PDi-1) was administered intraperitoneally (10 mg/kg, every 2 days, for a total of six doses) from day 15 to day 21. B . Tumor growth analysis across different groups. Left: Representative images of tumors from the indicated groups. Middle: Tumor weight comparison among groups. Right: Tumor volume progression over time. C . IHC staining for ASCC2, ALKBH3, E2F1, and G6PD in tumor tissues. Representative IHC images (left) and quantification of staining scores (right) for each marker. Scale bar = 50 μm. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ns, not significant

Article Snippet: The staining sequence and dye combination are as follows: DLEU1 probe ( NR_109973.1 , 5-1627, working solution, Alpha X Bio, China) - TSA520; E2F1 (1/200 dilution, Proteintech, Cat. #66515-1-Ig, RRID: AB_2881878) - TSA480; ALKBH3 (1/200 dilution, Proteintech, Cat. #12292-1-AP, RRID: AB_11125161) - TSA620; and ASCC2 (1/200 dilution, Proteintech, Cat.#11529-1-AP, RRID: AB_2059358) - TSA780.

Techniques: Knockdown, Comparison, Immunohistochemistry, Staining, Marker

Journal: Biomarker Research

Article Title: Histone modification-regulated LncRNA DLEU1 interacts with ASCC2/ALKBH3 complex to drive DNA repair, antioxidant homeostasis and glucose metabolism in gastric cancer

doi: 10.1186/s40364-025-00867-y

Figure Lengend Snippet: Expression and correlation analysis of DLEU1 and associated markers in normal and GC tissues, and its epigenetic regulation in GC. A . Representative multiplex immunofluorescence staining images showing DLEU1 (green), ASCC2 (white), ALKBH3 (orange), and E2F1 (cyan) in normal and tumor tissues ( N = 26, T = 104). Scale bar = 50 μm. B . Quantification of DLEU1 -positive cell density in normal and tumor tissues. C . Quantification of ASCC2-, ALKBH3-, and E2F1-positive cell densities in normal and tumor tissues. D . Percentage of E2F1 localization in the nucleus and cytoplasm in normal and tumor tissues. E . Scatter plots showing positive correlations between DLEU1 expression and ASCC2, ALKBH3, and E2F1 expression in tumor tissues. F . Correlation between DLEU1 expression and E2F1 localization in the cytoplasm and nucleus in tumor tissues. G . Correlation between ASCC2- and ALKBH3-positive cell in tumor tissues. H . HGC27 and MKN45 cells were treated with DMSO (control), Etoposide (10 µM, 24 h), 5-Fluorouracil (5-FU, 20 µg/mL, 24 h), or H 2 O 2 (10 µM, 2 h). The enrichment of H3K4me3 and H3K27ac at the DLEU1 promoter was analyzed by ChIP-qPCR. I . Proposed model illustrating how DLEU1 promotes gastric cancer progression via the ASCC2/ALKBH3/E2F1/G6PD axis, impacting transcriptional activation, DNA repair, and redox homeostasis. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ns, not significant

Article Snippet: The staining sequence and dye combination are as follows: DLEU1 probe ( NR_109973.1 , 5-1627, working solution, Alpha X Bio, China) - TSA520; E2F1 (1/200 dilution, Proteintech, Cat. #66515-1-Ig, RRID: AB_2881878) - TSA480; ALKBH3 (1/200 dilution, Proteintech, Cat. #12292-1-AP, RRID: AB_11125161) - TSA620; and ASCC2 (1/200 dilution, Proteintech, Cat.#11529-1-AP, RRID: AB_2059358) - TSA780.

Techniques: Expressing, Multiplex Assay, Immunofluorescence, Staining, Control, ChIP-qPCR, Activation Assay

Journal: Journal of Enzyme Inhibition and Medicinal Chemistry

Article Title: Targeting MED8 enhances sorafenib sensitivity in hepatocellular carcinoma by disrupting epithelial–mesenchymal transition mechanisms

doi: 10.1080/14756366.2025.2574988

Figure Lengend Snippet: MED8 regulates EMT through TRIP4. (A) MED8 interaction network from STRING website. (B) Mass spectrum showing unique peptides of TRIP4 identified by 2D-LC-MS/MS from protein lysates prepared from MHCC97H/SR cells after silencing MED8. (C–E) Representative western blot analysis of MED8 and TRIP4 protein expression in HCC and paired paracancerous tissues (n = 40, T, tumour; NT, non-tumour tissue). (F) Scatter plots showing a positive correlation between MED8 and TRIP4 protein expression levels in 40 HCC samples (n = 40, r = 0.3167, p = 0.0002, Pearson test). (G–I) Western blot analyses were used to detect MED8 and TRIP4 protein expression in cells stably transfected with the shMED8 or pcDNA-MED8 plasmid. (J, K) Western blot analysis confirming MED8 silencing and TRIP4 restoration and their effects on EMT-related biomarkers. (L) Western blot analysis showing the levels of MED8 overexpression and TRIP4 inhibition and their effects on EMT-related biomarkers.

Article Snippet: Sangon Biotech (Shanghai, China) synthesised shRNA-mediated RNA duplexes targeting MED8 and TRIP4.

Techniques: Liquid Chromatography with Mass Spectroscopy, Western Blot, Expressing, Stable Transfection, Transfection, Plasmid Preparation, Over Expression, Inhibition

Journal: Journal of Enzyme Inhibition and Medicinal Chemistry

Article Title: Targeting MED8 enhances sorafenib sensitivity in hepatocellular carcinoma by disrupting epithelial–mesenchymal transition mechanisms

doi: 10.1080/14756366.2025.2574988

Figure Lengend Snippet: MED8 interacts with TRIP4. (A) qRT-PCR analysis of MED8 and TRIP4 mRNA expression in HCC/SR. Left, HCCLM03/SR and MHCC97H/SR cells transfected with either shNC or shMED8. Right, HepG2/SR cells transfected with either vector or pcDNA-MED8. Data represent the mean ± SD of triplicate experiments and were statistically analysed with Student’s t-test; ns, not significant, **p < 0.01. (B) MHCC97H/SR and HCCLM03/SR cells were fixed and stained using TRIP4 (green) and MED8 (red) antibodies. Cell nuclei were stained using DAPI (blue). (C) Co-IP test was implemented to identify the protein binding of MED8 and TRIP4 in MHCC97H/SR cells. (D) Top ranked docking conformations and 3D structures of TRIP4 and MED8. TRIP4 and MED8 are displayed in cyan and green, respectively. (E) Diagram of TRIP4 truncated mutant constructs. (F) The N-terminal domain and the ASCH domain within the C-terminal region of TRIP4 interact with MED8. Mapping the TRIP4 domain that interacts with MED8. Constructs for MYC-tagged TRIP4 (full-length or truncated mutants) and FLAG-tagged MED8 were cotransfected into MHCC97H/SR cells. IP was performed using anti-MYC antibodies, and precipitates and input were analysed by western blotting with antibodies to FLAG and MYC.

Article Snippet: Sangon Biotech (Shanghai, China) synthesised shRNA-mediated RNA duplexes targeting MED8 and TRIP4.

Techniques: Quantitative RT-PCR, Expressing, Transfection, Plasmid Preparation, Staining, Co-Immunoprecipitation Assay, Protein Binding, Mutagenesis, Construct, Western Blot

Journal: Journal of Enzyme Inhibition and Medicinal Chemistry

Article Title: Targeting MED8 enhances sorafenib sensitivity in hepatocellular carcinoma by disrupting epithelial–mesenchymal transition mechanisms

doi: 10.1080/14756366.2025.2574988

Figure Lengend Snippet: MED8 stabilises TRIP4 protein expression by suppressing its ubiquitination and degradation. (A–C) TRIP4 protein levels at various times were measured by western blotting after MG132 addition (10 µM) to MHCC97H/SR, HCCLM03/SR, and HepG2/SR cells. (D–F) Western blot analysis of TRIP4 and MED8 protein expression in MHCC97H/SR and HCCLM03/SR cells transfected with shMED8-1# or shNC and HepG2/SR cells transfected with pcDNA-MED8 or control vector, with or without treatment with 10 µM MG132. (G–I) MHCC97H/SR and HCCLM03/SR cells transfected with shMED8-1# or shNC and HepG2/SR cells transfected with pcDNA-MED8 or control vector were subjected to treatment with 20 µg/mL CHX, followed by assessment of TRIP4 protein levels using western blotting. n = 3, **p < 0.01. (J–L) MHCC97H/SR, HCCLM03/SR, and HepG2/SR cells were treated with either shMED8-1# or pcDNA-MED8 for 72 hours, and intracellular proteasome activity was assessed using a proteasome activity fluorometric assay kit. The relative proteolytic activities are presented as average percentages of the total fluorescence intensity of the control group at the end of the assay. Data are shown as mean ± SD, n = 5, with statistical analysis performed using a two-sided Student’s t-test; ns, not significant. (M–O) Lysates of MHCC97H/SR, HCCLM03/SR, and HepG2/SR cells transfected with sh-MED8-1#, FLAG-MED8, and MYC-ubiquitin (MYC-Ub) were analysed by immunoblotting and then immunoprecipitated using anti-TRIP4, followed by probing with anti-MYC.

Article Snippet: Sangon Biotech (Shanghai, China) synthesised shRNA-mediated RNA duplexes targeting MED8 and TRIP4.

Techniques: Expressing, Ubiquitin Proteomics, Western Blot, Transfection, Control, Plasmid Preparation, Activity Assay, Fluorescence, Immunoprecipitation

Journal: Journal of Enzyme Inhibition and Medicinal Chemistry

Article Title: Targeting MED8 enhances sorafenib sensitivity in hepatocellular carcinoma by disrupting epithelial–mesenchymal transition mechanisms

doi: 10.1080/14756366.2025.2574988

Figure Lengend Snippet: Diagram of the regulatory mechanism of MED8 promoting sorafenib resistance in HCC. This is a proposed model by which MED8 inhibits the ubiquitination and degradation of TRIP4, thereby regulating its protein abundance, promoting EMT, and ultimately leading to sorafenib resistance in HCC cells.

Article Snippet: Sangon Biotech (Shanghai, China) synthesised shRNA-mediated RNA duplexes targeting MED8 and TRIP4.

Techniques: Ubiquitin Proteomics, Quantitative Proteomics