ro 3306  (Merck & Co)

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: 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