hydroxyurea  (Merck & Co)

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