dgtp solution  (New England Biolabs)

Journal: Journal of Translational Medicine

Article Title: PUF, a biflavone monomer, triggers DNA damage through SLC25A15 downregulation and purine metabolic suppression in DLBCL

doi: 10.1186/s12967-026-07797-9

Figure Lengend Snippet: PUF induces DNA damage in DLBCL cells. ( A ) effects of increasing concentrations of PUF on the expression of the DNA double-strand break marker γ‑H2AX in DLBCL cell lines, as assessed by Western blot. ( B ) quantitative densitometric analysis of the γ‑H2AX protein levels shown in ( A ). Data were normalized to GAPDH and are presented as mean ± SD ( n = 3). Statistical significance was determined by one‑way ANOVA followed by Dunnett’s test, using the 0 μg/mL PUF group as the control. * p < 0.05, ** p < 0.01, *** p < 0.001; ns, not significant. ( C ) Immunofluorescence staining of γ‑H2AX (green) and nuclei (DAPI, blue) in DLBCL cells treated with or without PUF. Scale bar, 100 μm. ( D ) Representative images from the comet assay showing DNA strand breaks in DLBCL cells after PUF treatment. DNA was stained red. Scale bar, 100 μm. ( E ) Western blot analysis showing the effect of co‑treatment with the purine synthesis intermediate IMP or the deoxyribonucleotide dGTP on PUF‑induced γ‑H2AX expression. ( F ) quantification of γ‑H2AX levels from ( E ). Data were normalized to GAPDH and are expressed as mean ± SD ( n = 3). One-way ANOVA with Dunnett’s test was performed using the group treated with PUF alone (0 μM IMP/dGTP) as the control. * p < 0.05, ** p < 0.01, *** p < 0.001; ns, not significant. ( G ) expression of γ‑H2AX in tumor tissues from xenograft mice treated with vehicle (Ctrl) or PUF (15 or 30 mg/kg). The blot was exposed for 10 s. ( H ) densitometric analysis of γ‑H2AX protein levels in ( G ). Data were normalized to β‑tubulin and are shown as mean ± SD ( n = 5 tumors/group). One‑way ANOVA with Dunnett’s test was used to compare each treatment group to the Ctrl group. * p < 0.05, ** p < 0.01, *** p < 0.001; ns, not significant

Article Snippet: Inosinic acid (MedChemExpress HY-108213) and deoxyguanosine triphosphate (trisodium) solution (MedChemExpress HY-W008661) were used.

Techniques: Expressing, Marker, Western Blot, Control, Immunofluorescence, Staining, Single Cell Gel Electrophoresis

Journal: Journal of Translational Medicine

Article Title: PUF, a biflavone monomer, triggers DNA damage through SLC25A15 downregulation and purine metabolic suppression in DLBCL

doi: 10.1186/s12967-026-07797-9

Figure Lengend Snippet: SLC25A15 knockdown phenocopies PUF-induced DNA damage. ( A ) in silico molecular docking predicts the direct binding of PUF to SLC25A15. The 3D representation shows PUF (yellow sticks) within the predicted binding pocket of SLC25A15 (gray surface). Key interacting residues are displayed. ( B ) cellular thermal shift assay (CETSA) analysis of the interaction between PUF and SLC25A15. Immunoblots show the thermal stability of SLC25A15 in SU-DHL-2 and OCI-LY8 cells treated with vehicle (DMSO) or PUF across a temperature gradient. ( C ) quantitative analysis of the CETSA data from ( B ). The percentage of remaining soluble SLC25A15 protein, normalized to β-tubulin, is plotted against temperature (mean ± SD, n = 3). A leftward shift in the melting curve upon PUF treatment indicates altered thermal stability due to ligand engagement. Significance compared to the 37 °C control group was determined by one-way ANOVA: * p < 0.05, ** p < 0.01, *** p < 0.001; ns, not significant. ( D ) schematic workflow for establishing SLC25A15 stable knockdown cell lines via lentiviral shRNA transfection and puromycin selection. ( E ) Western blot analysis of SLC25A15 and γ-H2AX expression in wild-type (WT) cells, negative control cells transfected with a non-targeting vector (NC), and three independent SLC25A15 knockdown clones (SH1, SH2, SH3). ( F ) quantitative densitometric analysis of the protein levels shown in ( E ). Data for γ-H2AX (red) and SLC25A15 (blue) were normalized to β-tubulin (mean ± SD, n = 3). Statistical significance compared to the NC group was determined by one-way ANOVA: * p < 0.05, ** p < 0.01, *** p < 0.001; ns, not significant. ( G ) Immunofluorescence staining for γ-H2AX (red) and nuclei (DAPI, blue) in SLC25A15 knockdown cells. Scale bar, 100 μm. ( H ) Immunofluorescence staining for SLC25A15 (red) and nuclei (DAPI, blue). Scale bar, 100 μm. ( I ) Western blot analysis showing the effect of supplementing dGTP on γ-H2AX and SLC25A15 levels in SLC25A15 knockdown (SH) cells compared to NC and WT cells after 48 hours of culture. ( J ) quantitative analysis of γ-H2AX (red) and SLC25A15 (blue) levels from ( I ), normalized to β-tubulin (mean ± SD, n = 3). For the SH group, statistical significance compared to the 0 μM dGTP condition was determined by one-way ANOVA: * p < 0.05, ** p < 0.01, *** p < 0.001; ns, not significant

Article Snippet: Inosinic acid (MedChemExpress HY-108213) and deoxyguanosine triphosphate (trisodium) solution (MedChemExpress HY-W008661) were used.

Techniques: Knockdown, In Silico, Binding Assay, Thermal Shift Assay, Western Blot, Control, shRNA, Transfection, Selection, Expressing, Negative Control, Plasmid Preparation, Clone Assay, Immunofluorescence, Staining