Journal: Neoplasia (New York, N.Y.)

Article Title: BPGM as an intrinsic brake to constrain metastasis through phospho-epigenetic-mediated carnitine biosynthesis suppression

doi: 10.1016/j.neo.2026.101299

Figure Lengend Snippet: 2,3-BPG-CDK1-EZH2-H3K27me3 Axis: BPGM’s epigenetic circuit breaker for cellular migration. (A) Integrated functional metabolomics analysis revealed BPGM-altered metabolites clustered in methyl donor group. Bubble size: metabolites count. (B) Hypothesis of molecular mechanism underlying BPGM regulated BBOX1 expression by post transcriptional modification (PTM). (C) Silencing BPGM significantly reduced the protein level of H3K27me3, while overexpressing BPGM increased its level. Cells stably expressing shBPGM/BPGM and its control cells (shCtrl/Ctrl) were used to detect protein level by western blotting. (D) ChIP assays disclosed that the fragments of BBOX1 and MMP9 promoter precipitated by anti-H3K27me3 antibody were increased upon overexpressing BPGM. SK-HEP-1 cells stably expressing BPGM and its control cells (Ctrl) were employed to ChIP assay. The antibody precipitated DNAs were amplified by qPCR. 5 % of the total DNAs were amplified to serve as the control for DNA content. Values shown are signal of α-H3K27me3-precipitated DNA relative to the input and the mean value of the control group was normalized as 1. (E) Overexpressing BPGM significantly increased the protein level of EZH2 but decreased the protein level of p-EZH2-T 345 in tumor cells. (F) The molecular docking of 2,3-BPG and CDK1. Predicted structure of 2,3-BPG binding with CDK1. Key contact residues: Thr14, Arg127, Arg170. (G) Overexpressing BPGM significantly increased the protein level of p-CDK1-T 14 in tumor cells. Cells stably expressing BPGM (BPGM-OE) and its control cells (Ctrl) were used to detect protein level by western blotting. (H) 2,3-BPG treatment enhanced the phosphorylation of CDK1 at thr14 in tumor cells. The indicated concentration of 2,3-BPG was incubated with the lysate of trophoblasts and tumor cells for 30 minutes followed by western blotting. (I-J) RO-3306 treatment enhanced the phosphorylation of CDK1 at thr14 and reduced the phosphorylation of EZH2 at thr345 in tumor cells. The tumor cells were treated with the indicated concentration of RO-3306 for 12 hours followed by western blotting. (K) The model deciphers the role of BPGM in regulating BBOX1 and MMP9 expression. Error bar: mean ± SEM. P -values are labeled above the bar chart.

Article Snippet: The following reagents were used: l -carnitine (541-15-1, Sigma-Aldrich, Saint Louis, MO, USA); 2,3-BPG (D5764, Sigma-Aldrich); CDK1 inhibitor, RO-3306 (S7747, Selleck, Shanghai, China).

Techniques: Migration, Functional Assay, Expressing, Modification, Stable Transfection, Control, Western Blot, Amplification, Binding Assay, Phospho-proteomics, Concentration Assay, Incubation, Labeling

Journal: Neoplasia (New York, N.Y.)

Article Title: BPGM as an intrinsic brake to constrain metastasis through phospho-epigenetic-mediated carnitine biosynthesis suppression

doi: 10.1016/j.neo.2026.101299

Figure Lengend Snippet: 2,3-BPG-CDK1-EZH2-H3K27me3 Axis: BPGM’s epigenetic circuit breaker for cellular migration. (A) Integrated functional metabolomics analysis revealed BPGM-altered metabolites clustered in methyl donor group. Bubble size: metabolites count. (B) Hypothesis of molecular mechanism underlying BPGM regulated BBOX1 expression by post transcriptional modification (PTM). (C) Silencing BPGM significantly reduced the protein level of H3K27me3, while overexpressing BPGM increased its level. Cells stably expressing shBPGM/BPGM and its control cells (shCtrl/Ctrl) were used to detect protein level by western blotting. (D) ChIP assays disclosed that the fragments of BBOX1 and MMP9 promoter precipitated by anti-H3K27me3 antibody were increased upon overexpressing BPGM. SK-HEP-1 cells stably expressing BPGM and its control cells (Ctrl) were employed to ChIP assay. The antibody precipitated DNAs were amplified by qPCR. 5 % of the total DNAs were amplified to serve as the control for DNA content. Values shown are signal of α-H3K27me3-precipitated DNA relative to the input and the mean value of the control group was normalized as 1. (E) Overexpressing BPGM significantly increased the protein level of EZH2 but decreased the protein level of p-EZH2-T 345 in tumor cells. (F) The molecular docking of 2,3-BPG and CDK1. Predicted structure of 2,3-BPG binding with CDK1. Key contact residues: Thr14, Arg127, Arg170. (G) Overexpressing BPGM significantly increased the protein level of p-CDK1-T 14 in tumor cells. Cells stably expressing BPGM (BPGM-OE) and its control cells (Ctrl) were used to detect protein level by western blotting. (H) 2,3-BPG treatment enhanced the phosphorylation of CDK1 at thr14 in tumor cells. The indicated concentration of 2,3-BPG was incubated with the lysate of trophoblasts and tumor cells for 30 minutes followed by western blotting. (I-J) RO-3306 treatment enhanced the phosphorylation of CDK1 at thr14 and reduced the phosphorylation of EZH2 at thr345 in tumor cells. The tumor cells were treated with the indicated concentration of RO-3306 for 12 hours followed by western blotting. (K) The model deciphers the role of BPGM in regulating BBOX1 and MMP9 expression. Error bar: mean ± SEM. P -values are labeled above the bar chart.

Article Snippet: The following reagents were used: l -carnitine (541-15-1, Sigma-Aldrich, Saint Louis, MO, USA); 2,3-BPG (D5764, Sigma-Aldrich); CDK1 inhibitor, RO-3306 (S7747, Selleck, Shanghai, China).

Techniques: Migration, Functional Assay, Expressing, Modification, Stable Transfection, Control, Western Blot, Amplification, Binding Assay, Phospho-proteomics, Concentration Assay, Incubation, Labeling

Journal: Journal of Biomedical Science

Article Title: IFI35 suppresses the transcription of hepatitis B virus cccDNA minichromosome via promoting HNF4α proteasomal degradation

doi: 10.1186/s12929-026-01239-w

Figure Lengend Snippet: IFI35 ubiquitinates HNF4a in TRIM21-dependent manner. A Cell lysates were immunoprecipitated using an anti-TRIM21 antibody. B Detection of HBV DNAs by Southern blot from HepG2 and HepG2-TRIM21-KO cells. Expression levels of HNF4α, IFI35 and TRIM21 in HepG2-TRIM21-KO cells compared to parental HepG2 cells (Control) were assessed by Western blot. HBV DNA levels of HepG2 and HepG2-TRIM21-KO cells were quantified in graph (bottom). C Quantification of HNF4α protein levels in HepG2 and HepG2-TRIM21 knockout (KO) cells transfected with increasing amounts of myc-IFI35. HNF4α levels were determined by immunoblotting and quantified relative to the vector control. Data represent mean ± SD from three independent experiments. D Secreted HBeAg and HBsAg levels were measured by ELISA in HepG2 and HepG2-TRIM21-KO cells transfected with increasing amounts of myc-IFI35. Data represent mean ± SD from three independent experiments. E HepG2 or HepG2-TRIM21 knockout cells were transfected with HBV 1.2mer together with increasing amounts of IFI35. Total RNA was analyzed by Northern blotting to detect HBV transcription. F Effect of TRIM21 on IFI35-mediated HNF4α degradation. HepG2 and HepG2-TRIM21-KO cells were transfected with either empty vector or myc-IFI35. After 48 h, cycloheximide (50ug/ml) were treated for the indicated times, and cells were harvested. Expression levels of HNF4α and IFI35 were evaluated through Western blotting. Quantification of HNF4α levels between HepG2 and HepG2-TRIM21-KO cells (bottom). G HepG2 cells and TRIM21 knockout cells were co-transfected with HA-tagged ubiquitin (1 μg) and either vector or IFI35 (2 μg). At 48 h post-transfection, HNF4α was immunoprecipitated and immunoblotted with anti-ubiquitin antibody to assess polyubiquitination. Total levels of HNF4α, IFI35, TRIM21, and HA-tagged ubiquitin in input lysates were also analyzed by immunoblotting. H Schematic diagram of HBV enhancer luciferase reporter constructs. Constructs containing enhancer I (EnhI), enhancer II (EnhII), or both regions were cloned upstream of the luciferase reporter gene. Mutant constructs lacking HNF4α binding sites were generated in EnhI (ΔHNF4α) or EnhII (ΔHNF4α1 and ΔHNF4α1,2) as indicated. I Basal luciferase activity of HBV enhancer reporter constructs. HepG2 cells were transfected with the indicated luciferase reporter plasmids, and luciferase activity was measured. J Comparison of the effect of IFI35 on EnhI-driven transcription with or without the HNF4α binding site. HepG2 cells were co-transfected with the indicated EnhI luciferase reporters and increasing amounts of myc-IFI35. Luciferase activity was measured 48 h post-transfection. Data represent mean ± SD from three independent experiments. K Comparison of the effect of IFI35 on EnhII-driven transcription with or without HNF4α binding sites. HepG2 cells were co-transfected with EnhII luciferase reporters containing wild-type or mutated HNF4α binding sites together with increasing amounts of myc-IFI35. Luciferase activity was measured 48 h post-transfection. Data represent mean ± SD from three independent experiments. The above experiment was independently conducted three times. Data are shown as mean ± SD. The statistical significance of the differences was assessed by the Student t test: *, P < 0.05; **, P < 0.01, ***, P < 0.001

Article Snippet: The following antibodies were used: IFI35 (sc-100769, Santa Cruz), HNF4α (H-171, Santa Cruz), HNF1α (Η-140, Santa Cruz), HNF3β (RY-7, Santa Cruz), core protein (B0586, Dako, Carpinteria, CA), TRIM21 (sc-25351, Santa Cruz), β-actin (A5441, Sigma) and GFP (sc-9996, Santa Cruz).

Techniques: Immunoprecipitation, Southern Blot, Expressing, Control, Western Blot, Knock-Out, Transfection, Plasmid Preparation, Enzyme-linked Immunosorbent Assay, Northern Blot, Ubiquitin Proteomics, Luciferase, Construct, Clone Assay, Mutagenesis, Binding Assay, Generated, Activity Assay, Comparison

Journal: Journal of Biomedical Science

Article Title: IFI35 suppresses the transcription of hepatitis B virus cccDNA minichromosome via promoting HNF4α proteasomal degradation

doi: 10.1186/s12929-026-01239-w

Figure Lengend Snippet: Domain mapping of IFI35 as mediators of TRIM21–HNF4α complex formation. A Schematic representation of IFI35 constructs used in this study. GFP-tagged mutants lacking NID1 (ΔNID1) or NID2 (ΔNID2), as well as the leucine zipper construct (Zip), were generated. B HepG2 cells were co-transfected with HBV 1.2mer and the indicated IFI35 constructs. At 72 h post-transfection, HBV replication intermediates were analyzed by Southern blotting. Quantification of HBV replication levels based on Southern blot analysis. C Secreted HBeAg and HBsAg levels in the culture supernatants were measured by ELISA. E Co-immunoprecipitation analysis of IFI35 mutants with HNF4α. HepG2 cells were transfected with GFP-tagged IFI35 constructs and cell lysates were immunoprecipitated using anti-HNF4α antibody followed by immunoblotting with anti-GFP antibody. F Interaction of IFI35 mutants with TRIM21. Cells were transfected with Myc-TRIM21 together with the indicated IFI35 constructs. Cell lysates were subjected to immunoprecipitation using anti-TRIM21 antibody followed by immunoblotting with anti-GFP antibody. Data represent mean ± SD (n = 3). Statistical significance was determined relative to the vector control

Article Snippet: The following antibodies were used: IFI35 (sc-100769, Santa Cruz), HNF4α (H-171, Santa Cruz), HNF1α (Η-140, Santa Cruz), HNF3β (RY-7, Santa Cruz), core protein (B0586, Dako, Carpinteria, CA), TRIM21 (sc-25351, Santa Cruz), β-actin (A5441, Sigma) and GFP (sc-9996, Santa Cruz).

Techniques: Construct, Generated, Transfection, Southern Blot, Enzyme-linked Immunosorbent Assay, Immunoprecipitation, Western Blot, Plasmid Preparation, Control

Journal: Communications Biology

Article Title: PACS1 syndrome mutation disrupts dynein-mediated cargo transport via HDAC6 and BICD2

doi: 10.1038/s42003-026-09924-0

Figure Lengend Snippet: a FLAG-HDAC6 co-expressed with BICD2–eGFP and HA-tagged PACS1 or PACS1 R203W was immunoprecipitated from HCT116 cells (M2 agarose); co-precipitating BICD2–eGFP and HA-tagged PACS1 variants detected by Western blot. Data are mean ± SEM, ANOVA with Dunnett’s post hoc, n = 3 independently normalized experiments. b Control (160) and patient (159) fibroblasts nucleofected with BICD2 or nonspecific (ns) siRNAs. After 48 h, cells were collected for Western blot ( bottom left ) or treated overnight with G1 inhibitor ribociclib (3 μM), G2/M inhibitor Ro-3306 (9 μM), or DMSO. Top: Cells were fixed, stained for Giantin (Golgi, red), α-tubulin (green), and DAPI (blue), then imaged by confocal microscopy. Scale bar, 20 μm. Bottom right : Quantification of Golgi dispersal (percentage of Giantin signal within or beyond 10 μm of nuclear envelope). Data are mean ± SEM, ANOVA with Dunnett’s post hoc, n > 50 cells per group from three independent experiments.

Article Snippet: Chemicals: DMSO (Sigma, 2650), ribociclib (Tocris, 7050), Ro-3306 (Tocris, 4181), rapalog (Takara Bio, 635057), and tubacin (Cayman Chemical, 13691-1).

Techniques: Immunoprecipitation, Western Blot, Control, Staining, Confocal Microscopy