Journal: Human Mutation

Article Title: Integrative Genomic and Functional Analysis Reveals NF1 Loss as a Modifier of DNA Damage and Replication Stress Responses in Ovarian Cancer

doi: 10.1155/humu/9333284

Figure Lengend Snippet: NF1 knockdown enhances replication stress sensitivity without altering basal proliferation in ovarian cancer cells. (a–b) Quantitative RT‐PCR showing efficient NF1 knockdown in (a) SK‐OV‐3 and (b) OVCAR‐3 cells expressing two independent NF1 shRNAs compared with control cells. (c–d) CCK‐8 assays showing comparable basal proliferation between NF1‐knockdown and control cells. (e–f) CCK‐8 assays following hydroxyurea treatment showing impaired growth of NF1‐deficient cells, indicating increased replication stress sensitivity. Data are presented as mean ± SEM from three independent experiments. Statistical significance was assessed using unpaired two‐tailed Student′s t ‐tests (∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001).

Article Snippet: To assess the effect of NF1 knockdown on sensitivity to replication stress, cells were treated with hydroxyurea (HU, 0.25 mM, MCE) 24 h after seeding.

Techniques: Knockdown, Quantitative RT-PCR, Expressing, Control, CCK-8 Assay, Two Tailed Test

Journal: The Journal of Biological Chemistry

Article Title: Interferon stimulatory DNA activates the DNA damage signaling through ATM and DNA-PK sensing

doi: 10.1016/j.jbc.2026.111362

Figure Lengend Snippet: Transfection of ISD leads to DNA damage signaling. A , HeLa and BJ-hTERT cells were transfected with 2 μg/ml of ISD (linear dsDNA) for 2, 4, 24 or 48 hours (h), then γH2AX was monitored by immunofluorescence. B , representative images of γH2AX staining ( red ) in HeLa cells transfected or not with 2 μg/ml ISD for 2, 4, 24 and 48 h. Hydroxyurea was used as a positive control. Nuclei were stained with DAPI ( blue ). The scale bar represents 100 μm. C , quantification of nuclear levels of γH2AX intensity (mean fluorescence intensity) is shown from a representative experiment in HeLa cells. Background levels were assessed performing the staining without primary (no Iary) or secondary antibodies (no IIary). Each dot shows the mean fluorescence intensity in an individual nucleus. The horizontal red line shows the median for each condition and is indicated. Around 100 nuclei or more were quantified in each condition. Non-parametric Mann and Whitney test was used to compare distributions and p -values are shown. D , HeLa cells were transfected for 24 h with 2 μg/ml of ISD and levels of γH2AX were quantified by fluorescent microscopy, as in C . Bars are the relative mean intensity (fold change) obtained from independent experiments. The relative fold change is indicated. Each dot is the mean relative intensity obtained from an independent experiment. p -values are the results of unpaired t test. E , quantification of nuclear levels of γH2AX is shown from a representative experiment in BJ-h TERT cells. Results are presented as in C. F , Western blot analysis of γH2AX in BJ-h TERT cells transfected or not with 2 μg/ml ISD. Non-specific band and Ponceau are shown as loading controls. Similar results were obtained in HeLa cells (shown in C ). G , BJ-hTERT cells were transfected or not with 2 μg/ml ISD for 24 h. p21/CDKN1A mRNA levels were quantified by RT–qPCR. Results are the mean of two independent experiments. The experiments shown are all representative of at least two independent experiments. γH2AX, phosphorylation of the histone H2AX; ISD, interferon stimulatory DNA

Article Snippet: Hydroxyurea (HU, Merck) was dissolved in water and used at final concentrations of 0.2 mM, 0.5 mM or 2 mM, as indicated in figures.

Techniques: Transfection, Immunofluorescence, Staining, Positive Control, Fluorescence, Microscopy, Western Blot, Quantitative RT-PCR, Phospho-proteomics

Journal: The Journal of Biological Chemistry

Article Title: Interferon stimulatory DNA activates the DNA damage signaling through ATM and DNA-PK sensing

doi: 10.1016/j.jbc.2026.111362

Figure Lengend Snippet: DNA-damage signaling induced by ISD is mediated by both ATM and DNA-PK kinases. A , BJ-hTERT cells were treated with 2 μM of ATM or DNA-PK inhibitors (i) 1 h prior to transfection with 2 μg/ml ISD for 24 h. Quantification of nuclear levels of γH2AX are shown as in C . Results are from one representative experiment (n = 2). B , the proliferation of BJ-h TERT fibroblasts was assessed for four consecutive days, following MOCK or ISD transfection. C , senescence-associated-β-galactosidase staining was performed 5 days after ISD transfection. Mean percentage and standard deviation for each condition is indicated (n = 2). A , B and C , hydroxyurea was used as positive control. One representative result is shown from at least two independent experiments. D , in BJ-hTERT cells, p21/CDKN1A mRNA levels were quantified by RT–qPCR in experimental conditions described in A (n = 2). E , our work suggests the following model. ISD, or other types of linear dsDNA, are sensed internally by cGAS and at DNA ends by DNA-PK and ATM. Activation of cGAS by foreign DNA leads to STING signaling and IFN-I response. In parallel to this DNA sensing, DNA-PK and ATM detect ISD and activate the DNA damage response. We also know that DNA-PK can constitute an alternative DNA sensing pathway, which results in IFN-I response (shown by an asterisk , References 9 and 20). Our results suggest that the consequence of activation of both cGAS- and DDR-signaling is cell proliferation arrest and senescence. See further details in the discussion. ATM, ataxia telangiectasia mutated; γH2AX, phosphorylation of the histone H2AX; ISD, interferon stimulatory DNA.

Article Snippet: Hydroxyurea (HU, Merck) was dissolved in water and used at final concentrations of 0.2 mM, 0.5 mM or 2 mM, as indicated in figures.

Techniques: Transfection, Staining, Standard Deviation, Positive Control, Quantitative RT-PCR, Activation Assay, Phospho-proteomics

Journal: bioRxiv

Article Title: Role of a childhood cancer-linked BRIP1/FANCJ germline variant in genomic instability and cancer cell vulnerability

doi: 10.64898/2026.03.24.714005

Figure Lengend Snippet: a, BRIP1 R162Q cells show increased sensitivity to ATR inhibition. Representative colony formation assays and quantification of HCT116 cells expressing BRIP1 WT, BRIP1 R162Q, or BRIP1 knockout (KO) treated with increasing doses of the ATR inhibitor VE-822. Cells were treated overnight and colonies were allowed to form for 10 days prior to crystal violet staining. b, Quantification of survival fraction following ATR inhibition by VE-822. c, BRIP1 R162Q expressing cells show increased sensitivity to DNA-PK inhibition. Representative colony formation assays and quantification following overnight treatment with increasing doses of the DNA-PK inhibitor NU7441. d, Quantification of survival fraction following DNA-PK inhibition using NU7441 e, BRIP1 R162Q cells show increased sensitivity to treatment with G4-stabilizer Pyridostatin. Representative colony formation assays following overnight treatment with increasing concentrations of Pyridostatin (PDS) are shown. After drug removal, cells were cultured for 10 days before fixation and staining. f, Quantification of surviving fraction following Pyridostatin treatment b,d,f , Quantification was performed using EagleEye analysis software. Data represent the mean ± SD of three independent biological replicates. g, Schematic representation of the workflow used to perform colony formation assays in BRIP1R162Q cells upon R-loop removal by ectopic RNaseH1 expression. BRIP1 R162Q cells were transfected with RNase H1 WT or catalytically inactive RNaseH1 D145N, re-seeded, and treated the next day with the drugs indicated. Colonies were stained with crystal violet after 10 days . h-k, RNaseH1 expression decreases sensitivity to ATR VE-822 ( h,i, ) and DNA-PK NU7441 ( j,k, ) inhibition in BRIP1 R162Q expressing cells. h,j, Show representative colony formation plates and i, k, show quantifications of clonogenic survival. Data represent mean values ± SEM from three biologically independent experiments. Colony formation was quantified using EagleEye analysis software.

Article Snippet: Cells were incubated with culture medium supplemented with mitomycin C (MMC; Carl Roth, 4150.1), hydroxyurea (HU; Sigma-Aldrich, H8627), pyridostatin (PDS; MedChemExpress, HY-15176), NU7441 (KU-57788; Selleckchem, S2638), or VE-822 (berzosertib; Selleckchem, S7102) at the indicated concentrations and time points.

Techniques: Inhibition, Expressing, Knock-Out, Staining, Cell Culture, Software, Transfection