Journal: Cell Death & Disease

Article Title: CHK1 is an integral regulator of DNA replication in human cells

doi: 10.1038/s41419-026-08624-1

Figure Lengend Snippet: A Schematic illustration of the strategy used to generate the dTAG-CHK1 knock-in cell line. FKBP12 F36V and an HA tag were inserted in frame at the endogenous CHEK1 locus using CRISPR/Cas9-mediated homologous recombination. B Genotyping PCR analysis confirming successful insertion of the dTAG cassette at the CHEK1 locus. C Immunoblot analysis showing time-dependent degradation of CHK1 protein following treatment with dTAG V -1 (1 μM). D Quantification of CHK1 protein levels from panel C, normalized to vinculin and plotted as the percentage of remaining CHK1 expression over time. E Representative images of colony formation assays. Wild-type (WT) and dTAG-CHK1 cells were treated with DMSO or dTAG V -1 for 6 days. F Cell viability of dTAG-CHK1 cells measured by a luminescence-based ATP assay over a 48-h time course in the presence or absence of dTAG V -1 ( n = 3). p < 0.05. G Schematic representation of CHK1 constructs used for rescue experiments, including full-length CHK1 (FL), full-length kinase-dead CHK1 (FLKD), N-terminal kinase domain (ND), kinase-dead N-terminal domain (NDKD), and ATR phosphorylation-deficient mutants (S317A/S345A and SQ/AQ). H Immunoblot analysis confirming expression of endogenous dTAG-CHK1 and exogenous SFB-tagged CHK1 WT and mutant constructs. I Colony formation assays in dTAG-CHK1 cells expressing the indicated CHK1 constructs and treated with DMSO or dTAG V -1.

Article Snippet: The CHK1 inhibitors rabusertib (LY2603618; MedChemExpress, HY-14720) and prexasertib (LY2606368; MedChemExpress, HY-18174), as well as the ATR inhibitor gartisertib (VX-803; MedChemExpress, HY-136270), were obtained from MedChemExpress.

Techniques: Knock-In, CRISPR, Homologous Recombination, Western Blot, Expressing, ATP Assay, Construct, Phospho-proteomics, Mutagenesis

Journal: Cell Death & Disease

Article Title: CHK1 is an integral regulator of DNA replication in human cells

doi: 10.1038/s41419-026-08624-1

Figure Lengend Snippet: A , B Neutral ( A ) and alkaline ( B ) comet assays showing DNA damage in dTAG-CHK1 cells treated with dTAG V -1 for 8 or 24 h. Quantification of comet tail moments is shown in the right panels. C Immunoblot analysis of DNA damage and replication stress markers in parental 293A and 293A-derived dTAG-CHK1 cells treated with dTAG V -1 or hydroxyurea (HU) for 2 or 24 h. D Time-course immunoblot analysis of dTAG-CHK1 cells treated with dTAG V -1 for the indicated durations, showing progressive accumulation of DNA damage markers and a reduction in pH3(S10) and cell-cycle-associated proteins. E EdU incorporation assays in parental 293A and 293A-derived dTAG-CHK1 cells treated with dTAG V -1. DNA synthesis was monitored at the indicated time points by EdU labeling and DAPI staining. F Flow cytometry analysis showing EdU incorporation and DNA content (FxCycle) in dTAG-CHK1 cells following dTAG V -1 treatment for the indicated durations.

Article Snippet: The CHK1 inhibitors rabusertib (LY2603618; MedChemExpress, HY-14720) and prexasertib (LY2606368; MedChemExpress, HY-18174), as well as the ATR inhibitor gartisertib (VX-803; MedChemExpress, HY-136270), were obtained from MedChemExpress.

Techniques: Western Blot, Derivative Assay, DNA Synthesis, Labeling, Staining, Flow Cytometry

Journal: Cell Death & Disease

Article Title: CHK1 is an integral regulator of DNA replication in human cells

doi: 10.1038/s41419-026-08624-1

Figure Lengend Snippet: A Schematic illustration of the synchronization and treatment protocol. dTAG-CHK1 cells were synchronized at the G1/S boundary using a double-thymidine block and released into early S phase for 2 h before treatment with dTAG V -1 or DMSO. B Cell viability analysis of synchronized dTAG-CHK1 cells following dTAG V -1 or DMSO treatment for the indicated durations, measured by a luminescence-based ATP assay ( n = 3). P < 0.05. C Immunoblot analysis showing induction of DNA damage and replication stress markers, as well as alterations in cell-cycle-associated proteins, following CHK1 depletion during S phase. D Cell-cycle analysis by flow cytometry (FxCycle versus EdU) showing S-phase progression and arrest in synchronized dTAG-CHK1 cells treated with DMSO or dTAG V -1.

Article Snippet: The CHK1 inhibitors rabusertib (LY2603618; MedChemExpress, HY-14720) and prexasertib (LY2606368; MedChemExpress, HY-18174), as well as the ATR inhibitor gartisertib (VX-803; MedChemExpress, HY-136270), were obtained from MedChemExpress.

Techniques: Blocking Assay, ATP Assay, Western Blot, Cell Cycle Assay, Flow Cytometry

Journal: Cell Death & Disease

Article Title: CHK1 is an integral regulator of DNA replication in human cells

doi: 10.1038/s41419-026-08624-1

Figure Lengend Snippet: A Schematic illustration of the synchronization and treatment strategy. Cells were synchronized at the G1/S boundary using a double-thymidine block and treated with DMSO or dTAG V -1 for the indicated durations without release into S phase. B Cell viability of G1/S-synchronized dTAG-CHK1 cells measured by a luminescence-based CellTiter-Glo assay following treatment with DMSO or dTAG V -1 ( n = 3). p < 0.05. C Immunoblot analysis showing induction of DNA damage and replication stress markers following CHK1 depletion in G1/S-arrested cells treated with dTAG V -1 for 0–8 h without release. D Flow cytometry analysis of EdU incorporation showing cell-cycle profiles of G1/S-synchronized dTAG-CHK1 cells treated with DMSO or dTAG V -1 for 0–8 h, demonstrating a lack of S-phase progression. E Cell viability analysis of G1/S-synchronized dTAG-MCM2 and dTAG-CHK1 cells treated with dTAG V -1 in the presence or absence of CDK2 inhibitor (CDK2i), CDC7 inhibitor (CDC7i), or combined treatment for 0-24 h. F Immunoblot analysis of DNA damage markers in G1/S-synchronized dTAG-MCM2 and dTAG-CHK1 cells treated with dTAG V -1 with or without CDK2i and/or CDC7i for 0–24 h.

Article Snippet: The CHK1 inhibitors rabusertib (LY2603618; MedChemExpress, HY-14720) and prexasertib (LY2606368; MedChemExpress, HY-18174), as well as the ATR inhibitor gartisertib (VX-803; MedChemExpress, HY-136270), were obtained from MedChemExpress.

Techniques: Blocking Assay, Glo Assay, Western Blot, Flow Cytometry

Journal: Cell Death & Disease

Article Title: CHK1 is an integral regulator of DNA replication in human cells

doi: 10.1038/s41419-026-08624-1

Figure Lengend Snippet: (1) During normal DNA replication, CHK1 ensures proper coupling between the CMG helicase and DNA polymerases through a feedback regulatory mechanism that maintains coordinated fork progression. (2) Moderate ATR-mediated activation of CHK1 is induced by partial uncoupling of the replicative helicase from DNA synthesis, which results in slowed CMG helicase progression to stabilize replication forks. (3) Strong ATR-mediated activation of CHK1 resulted from pronounced uncoupling of the replicative helicase from DNA synthesis, effectively halting CMG helicase activity to prevent replication-associated genome instability.

Article Snippet: The CHK1 inhibitors rabusertib (LY2603618; MedChemExpress, HY-14720) and prexasertib (LY2606368; MedChemExpress, HY-18174), as well as the ATR inhibitor gartisertib (VX-803; MedChemExpress, HY-136270), were obtained from MedChemExpress.

Techniques: Activation Assay, DNA Synthesis, Activity Assay

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: hnRNPA1-SF3B3 interaction drives radioresistance in oral squamous cell carcinoma by modulating MARF1 alternative splicing isoforms

doi: 10.1186/s13046-026-03697-4

Figure Lengend Snippet: hnRNPA1 promoted DNA repair through MARF1-L–mediated PPP1R10 mRNA degradation and activation of Chk1 phosphorylation. A - B Homologous recombination (HR) repair efficiency in OSCC cells transduced with scramble, sh-hnRNPA1, sh-hnRNPA1 + MARF1-L or sh-hnRNPA1 + MARF1-S constructs; ( C ) Representative immunofluorescence images and ( D )quantification assessing RAD51 foci formation in the indicated OSCC cell groups at 12 h after 6 Gy IR. Scale bar, 20 μm; ( E ) Enrichment analysis of Gene Ontology terms for downregulated differential genes (MARF1-L vs. MARF1-S groups), CC: Cellular Component, MF: Molecular Function. F Heatmap of qPCR analysis of PPP1R10 and PPP1R3G mRNA levels in control, MARF1-L, and MARF1-S OSCC cells. G Western blot analysis of PPP1R10, Chk1, p-Chk1 (S345) protein levels in the indicated OSCC cells at 12 h post-IR; ( H ) Schematic model illustrating how hnRNPA1 facilitated radioresistance by promoting MARF1-L–dependent degradation of PPP1R10 mRNA and subsequent activation of CHK1 signaling Statistical significance was determined by two-tailed Student’s t-test or two-way ANOVA followed by Tukey’s post hoc test, as appropriate. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001; ns, not significant

Article Snippet: OSCC cells were treated with the following pharmacological inhibitors: 25 nM of the PP1 inhibitor Okadaic acid (MCE, Cat. No. HY-N6785) for 24 h; 2 μM of the ATR inhibitor AZD6738 (MCE, Cat. No. AZD6738) for 24 h; and 1 μM of the CHK1 inhibitor MK8776 (MCE, Cat. No. MK8776) for 24 h.

Techniques: Activation Assay, Phospho-proteomics, Homologous Recombination, Transduction, Construct, Immunofluorescence, Control, Western Blot, Two Tailed Test

Journal: Nature Communications

Article Title: DNA-PK-mediated phosphorylation of STAT6 establishes a non-canonical type 2 immunity axis to prevent macrophage senescence

doi: 10.1038/s41467-026-69996-8

Figure Lengend Snippet: A Immunoblots of pSTAT6 (pY641) and γH2AX in thioglycolate-elicited peritoneal macrophages (Thio-PM) with Etoposide (Eto) and IL-4 treatment. The right panel shows the relative intensity of pSTAT6 (pY641) ( n = 4 independent experiments). B Immunofluorescence images of staining (pSTAT6 (pY641), red; DAPI, blue) of IL-4-stimulated Thio-PMs treated with Eto. Scale bars 50 μm. Right panel shows the quantification of pSTAT6 (pY641) ( n = 3 biological replicates per group). C Immunoblots of pSTAT6 (pY641) and γH2AX in Eto-treated Thio-PMs with different inhibitors. KU55933, an inhibitor of ATM. Berzosertib, inhibitor of ATR. NU7026, inhibitor of DNA-PK. AZD7762, inhibitor of CHK1/2. The right panel shows the quantification of pSTAT6 (pY641) ( n = 3 independent experiments). D Immunoblots of pSTAT6 (pY641) and γH2AX in Eto-treated Cas9 tg/+ Thio-PMs with indicated sgRNA. The right panel shows the quantification of pSTAT6 (pY641)/tSTAT6 ( n = 3 independent experiments). E Dual luciferase reporter of STAT6 in RAW 264.7 cells treated with Eto and NU7026 ( n = 3 per group). Data are mean ± s.e.m. p-value was calculated by paired two-tailed Student’s t test ( A , D ), unpaired two-tailed Student’s t test ( B ), one-way ANOVA with Dunnett’s correction ( C , E ).

Article Snippet: IL-4 (20 ng/mL, PeproTech, #214-14), DNA-PK inhibitor NU7026 (10 μM, Selleck Chemicals, #S2893), ATM inhibitor KU55933 (10 μM, Selleck Chemicals, #S1092), ATR inhibitor Berzosertib (0.5 μM, Selleck Chemicals, #S7102), CHK1/2 inhibitor AZD7762 (1 μM, Selleck Chemicals, #S1532), STAT6 inhibitor AS1517499 (2 μM, Selleck Chemicals, #S8685), STING inhibitor H151 (1 μM, Selleck Chemicals, #S6652), PTP1B inhibitor (2 μM, Santa Cruz, #sc-222227), PU.1 inhibitor DB2313 (10 nM, MedChem Express, #HY-124629), EPAC inhibitor ESI-09 (10 μM, Selleck Chemicals, #S7499), hIL-4 (20 ng/mL, PeproTech, #200-04), LPS (100 ng/mL, Sigma-Aldrich, #L2630), IFNγ (20 ng/mL, PeproTech, #315-05) were used.

Techniques: Western Blot, Immunofluorescence, Staining, Luciferase, Two Tailed Test

Journal: Oncology Research

Article Title: Extrachromosomal DNA in Solid Tumors—Landscape, Immune Effects, and Resistance to Targeted Therapy

doi: 10.32604/or.2026.075916

Figure Lengend Snippet: Pan-cancer representation of extrachromosomal DNA (ecDNA) and its biological and clinical effects. APOBEC3: Apolipoprotein B mRNA Editing Enzyme Catalytic Subunit 3, CCND1: Cyclin D1, ecDNA: Extrachromosomal DNA, lncRNAs: Long Non-Coding RNAs, ZNF330: Zinc Finger Protein 330, PITPNM3: Phosphatidylinositol Transfer Protein, Membrane-Associated 3, PDAC: Pancreatic Ductal Adenocarcinoma, MYC: MYC Proto-Oncogene, ABCB1: ATP-Binding Cassette Subfamily B Member 1, eccDNA: Extrachromosomal Circular DNA, SCARB1: Scavenger Receptor Class B Member 1, PDE10A: Phosphodiesterase 10A, HIF1A: Hypoxia-Inducible Factor 1 Alpha, HPV16: Human Papillomavirus Type 16, MDM2: Mouse Double Minute 2 Homolog, HMGA2: High Mobility Group AT-Hook 2, FGFR2: Fibroblast Growth Factor Receptor 2, ERBB2: Erb-B2 Receptor Tyrosine Kinase 2, RAB3B: RAB3B, Member RAS Oncogene Family, MYCN: MYCN Proto-Oncogene, CHK1: Checkpoint Kinase 1, CSF: Cerebrospinal Fluid.

Article Snippet: A potent and selective CHK1 inhibitor (BBI-cmpd1) exhibited enhanced biomarkers of replication stress and antiproliferative effects in ecDNA-positive cells compared to matched HSR or non-amplified controls.

Techniques: Membrane, Binding Assay

Journal: Oncology Research

Article Title: Extrachromosomal DNA in Solid Tumors—Landscape, Immune Effects, and Resistance to Targeted Therapy

doi: 10.32604/or.2026.075916

Figure Lengend Snippet: Mechanisms of extrachromosomal DNA (ecDNA)–driven resistance to targeted therapies across tumor models. KRAS: Kirsten Rat Sarcoma Viral Oncogene Homolog, ecDNA: Extrachromosomal DNA, MAPK: Mitogen-Activated Protein Kinase, FGFR2: Fibroblast Growth Factor Receptor 2, MYC: MYC Proto-Oncogene, AZD4547: AZD4547 (Selective Fibroblast Growth Factor Receptor Inhibitor), CHK1: Checkpoint Kinase 1, HSR: Homogeneous Staining Region, ABCG2: ATP-Binding Cassette Subfamily G Member 2, EGFR: Epidermal Growth Factor Receptor.

Article Snippet: A potent and selective CHK1 inhibitor (BBI-cmpd1) exhibited enhanced biomarkers of replication stress and antiproliferative effects in ecDNA-positive cells compared to matched HSR or non-amplified controls.

Techniques: Staining, Binding Assay