Journal: Cell Death & Disease

Article Title: DCTPP1 orchestrates dCTP pool dynamics and mtDNA stability in quiescent cells

doi: 10.1038/s41419-026-08632-1

Figure Lengend Snippet: A Time-course cell cycle profiling of CCD-34Lu cells subjected to quiescence induction by serum starvation (0.1% FBS) over the indicated days (left panel). Data points represent the mean of three biological replicates (mean ± S.D.). Statistical analysis was performed using two-way ANOVA (F (14, 30) = 100.80, p < 0.001), with Dunnett’s post hoc test for multiple comparisons. B Cell viability was determined using crystal violet live-cell staining in quiescent cells, expressed as the percentage of surviving attached cells. Tamoxifen (50 µM, 12 h) was used as a positive control for cell death. Results are shown as mean ± S.D. ( n ≥ 3), with individual data points indicated. Kruskal Wallis test with Dunn’s multiple comparison correction was applied for the statistical analysis. C Western blot analysis of pyrimidine metabolism enzymes in resting versus proliferating cells (upper panel). Only relevant sections of the full-length blots are shown; original blots are available in the Supplementary File. Quantification (lower panel) was performed using Fiji software, with band intensities normalized to the proliferative condition and α-tubulin used as a loading control. Data are presented as mean ± SD ( n ≥ 3), with individual data points shown. Statistical analysis was conducted using a two-tailed, unpaired t-test. D , E Left panel: Representative images showing the subcellular localization of DCTPP1 ( D ) or dUTPase ( E ) in proliferating and quiescent cells (cropped from 3D reconstructions). Mitochondria were stained with MitoTracker™ Red (red channel), DCTPP1 or dUTPase (green channel), and nuclei were stained with DAPI (blue channel). Scale bar; 10 µm. Right panel: Analysis of the percentage of relative co-localization of DCTPP1 ( D ) or dUTPase ( E ) with the nucleus (top) and mitochondria (bottom) presented as Manders coefficient. Co-localization was scored from ~30 cells per condition across three independent experiments ( n = 3), using Fiji software. Unless otherwise indicated, all experiments were carried out on day 10 of quiescence. For panels D and E, statistical analysis was performed using an unpaired two-tailed Student’s t-test.

Article Snippet: For these studies, we employed TH1217 (ZINC1775962367; HY-135909 MCE), a potent and selective DCTPP1 inhibitor with an IC50 of 47 nM [ ].

Techniques: Staining, Positive Control, Comparison, Western Blot, Software, Control, Two Tailed Test

Journal: Cell Death & Disease

Article Title: DCTPP1 orchestrates dCTP pool dynamics and mtDNA stability in quiescent cells

doi: 10.1038/s41419-026-08632-1

Figure Lengend Snippet: Pyrimidine nucleotides required for DNA synthesis (dCTP and dTTP) are produced through de novo and salvage pathways. In the de novo route, CDP and UDP are reduced to their deoxy forms by ribonucleotide reductase (RNR) and subsequently phosphorylated by nucleoside-diphosphate kinases to their triphosphate form. Formation of dTMP is catalysed by thymidylate synthase (TS) via methylation of dUMP, which mainly derives from dCMP deamination through deoxycytidylate deaminase (DCTD). Additional enzymes contribute to nucleotide catabolism: dUTP diphosphatase (dUTPase) hydrolyses dUTP to dUMP, and dCTP pyrophosphatase 1 (DCTPP1) converts dCTP to dCMP. In the salvage pathway, nucleosides released during nucleic acid turnover are phosphorylated by specific kinases: deoxycytidine kinase (dCK) for deoxycytidine and thymidine kinases, TK1 (cytosolic) and TK2 (mitochondrial) for uracil- and thymidine-derived nucleosides.

Article Snippet: For these studies, we employed TH1217 (ZINC1775962367; HY-135909 MCE), a potent and selective DCTPP1 inhibitor with an IC50 of 47 nM [ ].

Techniques: DNA Synthesis, Produced, Methylation, Derivative Assay

Journal: Cell Death & Disease

Article Title: DCTPP1 orchestrates dCTP pool dynamics and mtDNA stability in quiescent cells

doi: 10.1038/s41419-026-08632-1

Figure Lengend Snippet: A Cumulative proliferation growth curve of CCD-34Lu cells transfected with DCTPP1 (siDCTPP1) or control (siCtrl) siRNAs. Each data point represents the mean from three biological replicates, with values shown as mean ± S.D. ( n ≥ 3). A two-way ANOVA was performed (F (3,12) = 33.16; p < 0.001), followed by Dunnett’s multiple comparison correction. B Cell cycle profile of DCTPP1-siRNA transfected cells after 4 days of transfection, expressed as percentage of cells in each cell cycle phase. Values are mean ± S.D. ( n ≥ 3). A two-way ANOVA was performed with Sidak’s post hoc test to compare siDCTPP1 transfected cells (day 4) versus control cells (siCtrl), n.s. p < 0.12. C Western blot analysis of key pyrimidine metabolism enzymes in DCTPP1-depleted cells at the indicated time points (upper panel). Quantification of Western blot bands from siCtrl or siDCTPP1 cycling cells (lower panel). siRNA-transfected cells were used as controls, and anti-α-tubulin was employed to normalize protein levels. Band intensities were quantified using ImageJ/Fiji software. Only the relevant sections of the full-length blots are shown, with original blots provided in the Supplementary data. Values represent means ± S.D. ( n ≥ 3), individual data points shown. Statistical analysis was performed using the Kruskal–Wallis test, followed by Dunn’s multiple comparisons correction. D Immunofluorescence analysis of TK1 upon DCTPP1 down-regulation. Left panel, representative images of TK1 expression (green). Nuclei were stained with DAPI (blue) and mitochondria with MitoTracker™ Red (red). Scale bar, 10 µm; middle panel, quantification of total TK1 fluorescence intensity expressed as corrected total cell fluorescence (CTCF, x10 4 ) and distribution of fluorescence intensity levels as percentages (right panel), stratified into low (0-1000 a.u.), medium (1000-5000 a.u.) and high intensity (5000–10,000 a.u.). (Arbitrary Units, a.u.). Approximately 60 cells per condition were analysed across two independent experiments. Statistical analysis of TK1 CTCF was performed using an unpaired, two-tailed Mann–Whitney U test.

Article Snippet: For these studies, we employed TH1217 (ZINC1775962367; HY-135909 MCE), a potent and selective DCTPP1 inhibitor with an IC50 of 47 nM [ ].

Techniques: Transfection, Control, Comparison, Western Blot, Software, Immunofluorescence, Expressing, Staining, Fluorescence, Two Tailed Test, MANN-WHITNEY

Journal: Cell Death & Disease

Article Title: DCTPP1 orchestrates dCTP pool dynamics and mtDNA stability in quiescent cells

doi: 10.1038/s41419-026-08632-1

Figure Lengend Snippet: A Western blot analysis upon DCTPP1 depletion in CCD-34Lu quiescent by transfection with siDCTPP1 or siCtrl for 5 and 10 days, using 50 or 25 pmol of siRNA (right panel). Control siRNA-transfected cells were used as a control and anti-α-tubulin was used as a loading control. Western blot quantification (left panel). Values are mean ± S.D. ( n ≥ 3). B Crystal Violet assay assessing cell viability in DCTPP1 or control siRNA-transfected quiescent cells, with tamoxifen-treated cells (Tmxf, 50 µM 12 h) as positive control for cell death. Values are mean ± S.D. ( n ≥ 3). C Western blot comparison of pyrimidine metabolism enzyme expression levels in quiescent cells transfected with siCtrl or siDCTPP1, normalized to siCtrl-transfected cells and anti-α-tubulin were used as a loading control, at the indicated time points. Only relevant sections of full-length blots are shown, with original blots provided in Supplementary File. Results are shown as means ± SD ( n ≥ 3), with individual data points included. Statistical analysis was performed using the One-way ANOVA Kruskal Wallis test with Dunn’s multiple comparison correction for ( A–C ). D Representative high-resolution confocal microscopy images of the subcellular localisation of thymidylate synthase (TS, green) upon siCtrl (left panel) or siDCTPP1 transfection (right panel). Nuclei were stained with DAPI (blue), and mitochondria were stained with MitoTracker™ Red (red). 3D reconstructions are shown from frontal, basal, or apical views. Scale bar; 10 µm. E Quantification of total TS fluorescence intensity (corrected total cell fluorescence, CTCF) and TS mitochondrial colocalization (Manders coefficient) in approximately 30 cells scored per condition (siCtrl or siDCTPP1) using FIJI software (from three independent replicates; n = 3). Statistical analysis was performed using an unpaired two-tailed Student’s t -test.

Article Snippet: For these studies, we employed TH1217 (ZINC1775962367; HY-135909 MCE), a potent and selective DCTPP1 inhibitor with an IC50 of 47 nM [ ].

Techniques: Western Blot, Transfection, Control, Crystal Violet Assay, Positive Control, Comparison, Expressing, Confocal Microscopy, Staining, Fluorescence, Software, Two Tailed Test

Journal: Cell Death & Disease

Article Title: DCTPP1 orchestrates dCTP pool dynamics and mtDNA stability in quiescent cells

doi: 10.1038/s41419-026-08632-1

Figure Lengend Snippet: A Pyrimidine dNTP levels measured by the polymerase-based assay in DCTPP1-deficient proliferative cells upon 4 days post down-regulation and B in quiescent CCD-34Lu cells upon 10 days of DCTPP1 depletion. Data are presented as mean concentrations with error bars indicating ± SD from three independent experiments with triplicate technical replicates and individual values are shown. Statistical analyses in ( A ) and ( B ) were performed using a two-tailed Student’s t -test (confidence level 95%). C and D present a comparison of dNTP pools between proliferative and quiescent CCD-34Lu cells. In panel D, a two-way ANOVA followed by Sidak’s multiple comparison test was applied.

Article Snippet: For these studies, we employed TH1217 (ZINC1775962367; HY-135909 MCE), a potent and selective DCTPP1 inhibitor with an IC50 of 47 nM [ ].

Techniques: Two Tailed Test, Comparison

Journal: Cell Death & Disease

Article Title: DCTPP1 orchestrates dCTP pool dynamics and mtDNA stability in quiescent cells

doi: 10.1038/s41419-026-08632-1

Figure Lengend Snippet: A Mitochondrial function was evaluated using dual staining with MitoTracker™ Green (mitochondrial mass) and MitoTracker™ Red (membrane potential), followed by flow cytometry analysis. Data are expressed as MFI-corrected median fluorescence intensity (MFI), in arbitrary units (a.u., log10 scale), across all experimental conditions. Error bars represent standard deviation (S.D.) from three biological replicates, with cells cultured in separate wells/dishes within a single experiment. B Mitochondrial membrane potential was normalized and expressed as a percentage by calculating the ratio of fluorescence intensity of MitoTracker™ Red (membrane potential-sensitive dye) to that of MitoTracker™ Green. Data are presented as means ± S.D. ( n ≥ 3), with individual data points included. A two-way ANOVA was performed, followed by Tukey’s post hoc test, revealing a significant interaction effect (F (2,14) = 36.40, p < 0.001). C mtDNA copy number was determined by real-time quantitative PCR, measuring the relative mitochondrial DNA content (mtDNA/nDNA) under various conditions, including proliferation versus quiescence and siRNA depletion (siCtrl vs. siDCTPP1) in both proliferation and quiescence states, at the indicated time points. Individual data points are shown ( n ≥ 6). D mtDNA copy number normalized to citrate synthase (CS) activity (nmol/min × mg protein, see Supplementary Fig. ) is shown, with each column representing the mean of (mtDNA/nDNA)/CS values ± S.D. ( n ≥ 3). Statistical significance was assessed using an unpaired Student’s t -test for ( C ) and ( D ) (left; proliferation vs. quiescence), and a Kruskal–Wallis test followed by Dunn’s multiple comparisons correction for the right panels in ( C ) and ( D ) (siCtrl vs. siDCTPP1 in proliferation and quiescence, respectively). E Representative in situ images of mtDNA in quiescent cells transfected with either control (siCtrl) or DCTPP1-targeted (siDCTPP1) siRNA for 10 days. Nuclei were stained with DAPI (blue), mitochondrial signal was marked with MitoTracker™ Red (red), and in situ mtDNA-positive dots are shown in grey (far-red). A mask was applied to quantify mtDNA-positive dots, as shown in the confocal images. Left panel: Quantification of mtDNA-positive dots using Fiji software, with individual data points presented ( n ≥ 30). Statistical analysis was performed using an unpaired, two-tailed Mann–Whitney U test. Scale bar: 10 µm.

Article Snippet: For these studies, we employed TH1217 (ZINC1775962367; HY-135909 MCE), a potent and selective DCTPP1 inhibitor with an IC50 of 47 nM [ ].

Techniques: Staining, Membrane, Flow Cytometry, Fluorescence, Standard Deviation, Cell Culture, Real-time Polymerase Chain Reaction, Activity Assay, In Situ, Transfection, Control, Software, Two Tailed Test, MANN-WHITNEY

Journal: Cell Death & Disease

Article Title: DCTPP1 orchestrates dCTP pool dynamics and mtDNA stability in quiescent cells

doi: 10.1038/s41419-026-08632-1

Figure Lengend Snippet: A DCTPP1 western blot analysis was performed in CCD-34Lu quiescent cells after thymidine overload at the indicated dThd concentrations (0-160 μM). Western blot quantification was performed using Fiji software and α-tubulin as loading control (right panel). Original blots are presented in Supplementary data. B Representative immunofluorescence images of DCTPP1 staining in the presence or absence of dThd overload are shown (left panel). Showing nuclei stained with DAPI (blue channel), mitochondrial signal labeled with MitoTracker™ Red (red channel), and DCTPP1 staining (green channel). C mtDNA copy number was quantified by qPCR after dThd overload (40 µM) at the indicated time points. The mtDNA copy number (mtDNA/nDNA) was normalized to control conditions without dThd. Data are presented as mean ± SD ( n ≥ 3). Statistical analysis was performed using a Kruskal–Wallis test, followed by Dunn’s multiple comparisons correction for ( A ) and ( C ). D Reversal of dThd overload-induced mtDNA defects was assessed after dCyt (40 µM) administration, measured by qPCR at indicated times. Each point represents the mean from three biological replicates ( n = 3). Statistical analysis was conducted using a two-way ANOVA (F (6, 60) = 45.02, p < 0.001), with Dunnett’s post hoc test applied for multiple comparisons. E Representative immunofluorescence images of mtDNA-positive dots (left panel) are shown; red channel: mitochondria marker (MitoTracker™ Red), blue channel: nucleus staining (DAPI), and grey (far-red channel): mtDNA staining with anti-DNA antibody, clone AC-30-10 (CBL186), in the presence or absence of dThd (40 µM). Right panel: Quantification of mtDNA-positive dots was performed using Fiji software on 30 individual cells per condition, across three independent experiments ( n = 3). Statistical analysis was conducted using an unpaired, two-tailed Student’s t-test. Scale bar; 10 µm.

Article Snippet: For these studies, we employed TH1217 (ZINC1775962367; HY-135909 MCE), a potent and selective DCTPP1 inhibitor with an IC50 of 47 nM [ ].

Techniques: Western Blot, Software, Control, Immunofluorescence, Staining, Labeling, Marker, Two Tailed Test

Journal: Cell Death & Disease

Article Title: DCTPP1 orchestrates dCTP pool dynamics and mtDNA stability in quiescent cells

doi: 10.1038/s41419-026-08632-1

Figure Lengend Snippet: A mtDNA copy number was determined by real-time quantitative PCR, measuring the relative mitochondrial DNA content (mtDNA/nDNA) upon DCTPP1 depletion by siRNA in presence or absence of thymidine overload (40 µM dThd) at indicated time points. Control siRNA-transfected cells in absence of dThd overload were used as a control. Individual data points are shown ( n ≥ 6), with statistical significance determined using a two-way ANOVA followed by Tukey’s multiple comparisons correction. B Representative immunofluorescence images of mtDNA-positive dots (left panel) following DCTPP1 depletion, in the presence or absence of dThd overload. Control siRNA-transfected cells are included as internal controls. Mitochondria are labeled with MitoTracker™ Red (red channel), nuclei are stained with DAPI (blue channel), and mtDNA is visualized in grey using an anti-DNA antibody (clone AC-30-10, CBL186; far-red channel). dThd was used at 40 µM. Scale bar; 10 µm. Right panel: Quantification of mtDNA-positive dots was performed using Fiji software ( ≥ 30 cells per condition from three independent experiments, n = 3). C dNTP pools alteration, particularly dCTP (left panel) and dTTP (right panel), in response to thymidine overload was analized. Cells were transient silencing for Control or DCTPP1-target siRNA during 20 days and dNTP levels were measured by a polymerase-based assay. Data are presented as mean concentrations, with error bars representing mean ± SD from three independent experiments with triplicate technical replicates. Individual values are also shown. Statistical analysis was performed using an unpaired two-tailed Student’s t -test for ( B , C ).

Article Snippet: For these studies, we employed TH1217 (ZINC1775962367; HY-135909 MCE), a potent and selective DCTPP1 inhibitor with an IC50 of 47 nM [ ].

Techniques: Real-time Polymerase Chain Reaction, Control, Transfection, Immunofluorescence, Labeling, Staining, Software, Two Tailed Test

Journal: Cell Death & Disease

Article Title: DCTPP1 orchestrates dCTP pool dynamics and mtDNA stability in quiescent cells

doi: 10.1038/s41419-026-08632-1

Figure Lengend Snippet: A Cytotoxicity profile of the DCTPP1 inhibitor TH1217 in quiescent CCD-34Lu fibroblasts over 10–20 days at concentrations ranging from 0.5–10 µM. Tamoxifen (50 µM, Tmxf) was used as a positive toxicity control. Statistical analysis was conducted using a two-way ANOVA (F (11, 59) = 2.55, p = 0.010), with Dunnett’s post hoc test applied for multiple comparisons. B mtDNA copy number in CCD-34Lu cells subjected to dThd overload (40 μM, 20 days) with or without TH1217 (0.05–2.5 µM), showing rescue of mtDNA depletion. Ordinary one-way ANOVA with Dunnett’s post hoc test was applied for statistical analysis of the effect of TH1217 treatment in thymidine treated cells while the effect of thymidine overloading alone on mtDNA versus non-treated controls was analysed using the Student’s t-test C dCTP (left panel) and dTTP (right panel) pools in response to 20 days of TH1217 treatment (2.5 μM) in the presence or absence of thymidine overload (40 μM, 20 days) was analysed. Data are presented as mean concentrations, with error bars representing mean ± SD from three independent experiments. Individual values are also shown. Statistical analysis was performed using an unpaired two-tailed Student’s t -test.

Article Snippet: For these studies, we employed TH1217 (ZINC1775962367; HY-135909 MCE), a potent and selective DCTPP1 inhibitor with an IC50 of 47 nM [ ].

Techniques: Control, Two Tailed Test

Journal: Frontiers in Oncology

Article Title: Spatial transcriptome and single-cell sequencing reveal the role of nucleotide metabolism in breast cancer progression and tumor microenvironment

doi: 10.3389/fonc.2025.1703778

Figure Lengend Snippet: Predicting therapy response with NMRS and validation of the core genes. (A) Estimated IC50 values of Tamoxifen and Palbociclib in high- versus low NMRS groups. (B) Estimated IC50 values of Olaparib and Gefitinib in high- versus low NMRS groups. (C, D) The correlation and differential analysis of drug sensitivity for potential drugs screened from the CTRP and PRISM datasets. (E) Validation of the expression of TAGLN2, PCMT1, PTMA, TUBA3D, and DCTPP1 in a normal breast cell line (MCF-10A) and three BC cell lines (MDA-MB-231, MCF7, and SK-BR3) by qRT-PCR. (F) Immunohistochemical images showing the protein expression of the five genes in the HPA database. **** p < 0.0001, *** p < 0.001, ** p < 0.01, * p < 0.05, ns p > 0.05. BC breast cancer, HPA Human Protein Atlas.

Article Snippet: The antibodies used in this study include: DCTPP1 (Proteintech, # 16684-1-AP, 1:1000), β-Actin (Proteintech, #66009-1-Ig, 1:5000) and anti-Rabbit secondary antibody (Proteintech, Cat: #SA00001-2, 1:5000).

Techniques: Biomarker Discovery, Expressing, Quantitative RT-PCR, Immunohistochemical staining

Journal: Frontiers in Oncology

Article Title: Spatial transcriptome and single-cell sequencing reveal the role of nucleotide metabolism in breast cancer progression and tumor microenvironment

doi: 10.3389/fonc.2025.1703778

Figure Lengend Snippet: DCTPP1 expression and functional validation in BC (A) DCTPP1 mRNA expression was higher in TCGA-BRCA tumor tissues than in normal tissues ( p < 0.01). (B) Overall survival was worse in TCGA-BRCA patients with high DCTPP1 expression ( p < 0.0001). (C) ROC curve analysis of DCTPP1, highlighting its diagnostic potential. (D) Single-cell analysis showed ( GSE161529 ) that DCTPP1 expression was significantly higher in NUhighepi cells than in others. (E, F) WB and qpcr analysis of the knockdown efficiency of DCTPP1 in MDA-MB-231 (E) and SK-BR-3 (F) cells and showed that the DCTPP1 gene was successfully knocked down in MDA-MB-231 and SK-BR-3 cell lines. (G) CCK-8 showed that the proliferation activity of the cells that knockdown DCTPP1 was dramatically reduced. (H) After DCTPP1 knockdown, the cloning ability of MDA-MB-231 and SK-BR-3 cell lines decreased significantly. (I) Healing test. After DCTPP1 knockdown, the migration ability of MDA-MB-231 and SK-BR-3 cell lines decreased significantly. (J) Transwell assay. After DCTPP1 knockdown, the migration and invasion abilities of MDA-MB-231 and SK-BR-3 cell lines were significantly decreased. **** p < 0.0001, *** p < 0.001, * p < 0.05, ns p > 0.05.

Article Snippet: The antibodies used in this study include: DCTPP1 (Proteintech, # 16684-1-AP, 1:1000), β-Actin (Proteintech, #66009-1-Ig, 1:5000) and anti-Rabbit secondary antibody (Proteintech, Cat: #SA00001-2, 1:5000).

Techniques: Expressing, Functional Assay, Biomarker Discovery, Diagnostic Assay, Single-cell Analysis, Knockdown, CCK-8 Assay, Activity Assay, Cloning, Migration, Transwell Assay