atr inhibitor  (TargetMol)

Journal: PLOS Biology

Article Title: DNA damage induced by HIV-1 Vpr triggers epigenetic remodeling and transcriptional programs to enhance virus transcription and latency reactivation

doi: 10.1371/journal.pbio.3003621

Figure Lengend Snippet: (A) Immunofluorescence microscopy quantification of DDR activation and histone marks in HeLa cells infected with control or Vpr-expressing virus. Vpr-infected HeLa cells were infected for 24 hours and then treated with vehicle or 3 mM caffeine for 24 hours ( n = 50 cells). Analyses performed using a one-way ANOVA; ns, not significant; *** p < 0.001. The data underlying this Figure can be found in . (B) Immunofluorescence microscopy quantification of histone marks in HeLa cells infected with control or Vpr-expressing virus in the presence or absence of vehicle, 3 mM caffeine, 10 nM ATM i , or 10 μM ATR i ( n = 50 cells). Cells were infected for 24 hours prior to inhibitor treatment for 24 hours and preparation for immunofluorescence microscopy. Analyses performed using a one-way ANOVA; ns, not significant; *** p < 0.001. The data underlying this Figure can be found in . (C) Immunofluorescence microscopy quantification of histone marks in HeLa cells infected with control or virus expressing the indicated Vpr subtype consensus sequence. Cells were infected for 24 hours and then treated with vehicle or with 3mM caffeine for 24 hours prior to preparation for immunofluorescence microscopy ( n = 50 cells). Analyses performed using a one-way ANOVA; ns, not significant; *** p < 0.001. The data underlying this Figure can be found in . (D) Cell cycle analysis of DDR activation and histone marks in HeLa cells infected with control or Vpr-expressing viruses. Left, flow cytometric analysis of propidium iodide (PI) stained HeLa or MDM cells infected with the indicated virus. Right, flow cytometric analysis of immunolabeled and PI-stained HeLa cells infected with the indicated virus ( n = 4 experiment). Analyses performed using a one-way ANOVA; ns, not significant; *** p < 0.001; ** p < 0.01. The data underlying this Figure can be found in . Representative gating strategies are depicted in and raw FSC files can be found in the Figshare Data repository ( https://doi.org/10.6084/m9.figshare.c.8239897 ).

Article Snippet: For inhibitor experiments, cells were treated 24-hours post-infection with either 10 nM ATM inhibitor (Fisher, #AZD1390), 10 μM ATR inhibitor (Fisher, #NU6027), 3 mM caffeine, 16 μM DNA-PK inhibitor #1 (MedChemExpress, #NU7026), or 16 μM DNA-PK inhibitor #2 (MedChemExpress #NU7441) for 24 hours.

Techniques: Immunofluorescence, Microscopy, Activation Assay, Infection, Control, Expressing, Virus, Sequencing, Cell Cycle Assay, Staining, Immunolabeling

Journal: PLOS Biology

Article Title: DNA damage induced by HIV-1 Vpr triggers epigenetic remodeling and transcriptional programs to enhance virus transcription and latency reactivation

doi: 10.1371/journal.pbio.3003621

Figure Lengend Snippet: (A) Top, schematic of proviruses used in these experiments. For these constructs, mCherry expression is driven off of the HIV promoter as opposed to being driven off of CMV. Bottom, flow cytometric analysis of HIV LTR activity in THP1 or HeLa cells infected with increasing MOI of the indicated viruses ( n = 3 experiments). For inhibitor treatments, cells were infected for 24 hours prior to a 24-hour treatment with 3 mM caffeine or combined ATM (10 nM)/ATR (10 μM) treatment. ns, not significant; * p < 0.05; ** p < 0.01; *** p < 0.001. The data underlying this Figure can be found in . Representative gating strategies are depicted in and raw FSC files can be found in the Figshare Data repository ( https://doi.org/10.6084/m9.figshare.c.8239897 ). (B) Flow cytometric quantification of HIV LTR activity in primary MDMs infected with Vpr WT virus in the presence or absence of DDR inhibition ( n = 7 experiments). MDMs were infected for 24 hours prior to treating with combined ATM (10 nM)/ATR (10 μM) inhibitors for 24 hours. Cells were detached and mCherry fluorescence was quantified via flow cytometry. ** p < 0.01. The data underlying this Figure can be found in . Representative gating strategies are depicted in and raw FSC files can be found in the Figshare Data repository ( https://doi.org/10.6084/m9.figshare.c.8239897 ). (C) Flow cytometric quantification of LTR-mCh MFI in infected HeLa cells subjected to ATM (10nM), ATR (10 μM), or combined ATM and ATR inhibition 48 hours post-infection (n = 5 experiments). Cells were detached and mCherry fluorescence was quantified via flow cytometry. ns, not significant. The data underlying this Figure can be found in . Representative gating strategies are depicted in and raw FSC files can be found in the Figshare Data repository ( https://doi.org/10.6084/m9.figshare.c.8239897 ). (D) Top, schematic of proviruses used in these experiments. For these proviruses, BFP was incorporated into the gag gene to generate Gag-BFP proteins from the viral promoter. Bottom left, live cell fluorescence microscopy images of HeLa cells 48-hours post-co-transfection with the indicated Gag-BFP proviruses and an eGFP control plasmid. Bottom middle, flow cytometric histogram of BFP fluorescence in eGFP-positive HeLa cells. Bottom right, quantification of BFP MFI in eGFP-positive cells with or without DDR inhibition ( n = 3 experiments). Cells were co-transfected for 24 hours prior to adding the indicated inhibitor for 24 hours. Analyses performed using a one-way ANOVA; ns, not significant; *** p < 0.001; * p < 0.05. The data underlying this Figure can be found in . Representative gating strategies are depicted in and raw FSC files can be found in the Figshare Data repository ( https://doi.org/10.6084/m9.figshare.c.8239897 ). (E) Diagram displaying the establishment of the HeLa latency model H-Lats (left) and representative live cell fluorescence microscopy images of H-Lat GFP expression when infected with indicated viruses. H-Lat cells were infected for 48 hours prior to being subjected to live cell fluorescence microscopy. Right, flow cytometric analysis of H-Lat GFP expression 48 hours post-infection with the indicated viruses. (F) Flow cytometric analysis of H-Lat reactivation following infection with increasing MOI of the indicated viruses in the presence or absence of DDR inhibition ( n = 3 experiments). Cells were infected for 24 hours prior to inhibitor treatment for 24 hours and preparation for flow cytometry. *** p < 0.001. The data underlying this Figure can be found in . Representative gating strategies are depicted in and raw FSC files can be found in the Figshare Data repository ( https://doi.org/10.6084/m9.figshare.c.8239897 ). (G) Flow cytometric quantification of LTR-eGFP MFI in infected H-Lat cells subjected to ATM (10nM), ATR (10 μM), or combined ATM/ATR treatment. H-Lat cells were infected for 24 hours prior to a 24-hour inhibitor treatment and preparation for flow cytometry ( n = 5 experiments). ns, not significant; * p < 0.05. The data underlying this Figure can be found in . Representative gating strategies are depicted in and raw FSC files can be found in the Figshare Data repository ( https://doi.org/10.6084/m9.figshare.c.8239897 ). (H) Flow cytometric analysis of CEM-GFP, J-Lat clone 10.6, and monocyte clone U1 latency models uninfected or infected with Vpr WT virus in the presence or absence of 3 mM caffeine ( n = 3 experiments). Cells were infected with increasing MOI of Vpr-expressing virus for 24 hours, treated with vehicle or 3 mM caffeine for 24 hours, and then subjected to flow cytometry. * p < 0.05; ** p < 0.01; *** p < 0.001. The data underlying this Figure can be found in . Representative gating strategies are depicted in and raw FSC files can be found in the Figshare Data repository ( https://doi.org/10.6084/m9.figshare.c.8239897 ). (I) Flow cytometric quantification of LTR-eGFP MFI in H-Lat cells transiently expressing plasmids encoding Tat and/or Vpr and treated with 50 µM etoposide or vehicle ( n = 3 experiments). H-Lats were transfected with the indicated plasmid combination for 24 hours, treated with vehicle or etoposide for 24 hours, and then subjected to flow cytometry. Analyses performed using a one-way ANOVA; ** p < 0.01. The data underlying this Figure can be found in . Representative gating strategies are depicted in and raw FSC files can be found in the Figshare Data repository ( https://doi.org/10.6084/m9.figshare.c.8239897 ).

Article Snippet: For inhibitor experiments, cells were treated 24-hours post-infection with either 10 nM ATM inhibitor (Fisher, #AZD1390), 10 μM ATR inhibitor (Fisher, #NU6027), 3 mM caffeine, 16 μM DNA-PK inhibitor #1 (MedChemExpress, #NU7026), or 16 μM DNA-PK inhibitor #2 (MedChemExpress #NU7441) for 24 hours.

Techniques: Construct, Expressing, Activity Assay, Infection, Virus, Inhibition, Fluorescence, Flow Cytometry, Microscopy, Cotransfection, Control, Plasmid Preparation, Transfection

Journal: PLOS Biology

Article Title: DNA damage induced by HIV-1 Vpr triggers epigenetic remodeling and transcriptional programs to enhance virus transcription and latency reactivation

doi: 10.1371/journal.pbio.3003621

Figure Lengend Snippet: (A) Immunofluorescence microscopy quantification of NFκB translocation, phosphorylated SP1 residue Ser101, phosphorylated cJun residues Ser63 and Ser73, and phosphorylated RNA polymerase II residues Ser2 and Ser5 in differentiated THP1 cells infected with Vpr WT or control viruses in the presence or absence of ATM, ATR, or DNA-PK inhibition ( n = 50 cells). Cells were infected for 24 hours, treated with vehicle or the indicated inhibitor for 24 hours, and then subjected to immunofluorescence microscopy. Analyses were performed using a one-way ANOVA; ns, not significant; ** p < 0.01; *** p < 0.001. The data underlying this Figure can be found in . (B) Representative immunofluorescence microscopy images of R-loop abundance in primary MDM and HeLa cells infected with indicated viruses 48 hours post-infection ( n = 50 cells). Analyses performed using a student t test; *** p < 0.001. (C–E) Immunofluorescence microscopy quantification of R-loop abundance in HeLa cells infected with indicated viruses 48 hours post-infection. Samples were left untreated (C) or treated with RNaseH (D, left), triptolide (D, right), or with the indicated DDR inhibitors (E), respectively ( n = 50 cells). For RNaseH treatment, cells were infected for 48 hours prior to methanol fixation and addition of recombinant RNaseH to deplete RNA associated with R-loops. For inhibitor treatments, HeLa cells were infected for 24 hours, treated with the indicated inhibitor for 24 hours, and then subjected to immunofluorescence microscopy. Analyses were performed using a one-way ANOVA; ns, not significant; ** p < 0.01; *** p < 0.001. The data underlying this Figure can be found in . (F) Immunofluorescence microscopy quantification of DDR activation (left) and R-loop abundance (right) in HeLa cells infected with indicated viruses and transfected with RNaseH constructs ( n = 50 cells). Cells were infected for 24 hours before transfection of control or RNaseH-expressing plasmids for 24 hours prior to being prepared for immunofluorescence microscopy. Analyses performed using a one-way ANOVA; ns, not significant; *** p < 0.001; * p < 0.05. The data underlying this Figure can be found in . (G) Flow cytometric histograms of HeLa cells infected with control or Vpr WT LTR-mCh viruses for 24 hours prior to transient expression of RNaseH for 24 hours and quantification of LTR-mCh MFI in transfected cells via flow cytometry ( n = 3 experiments). Analyses performed using a one-way ANOVA; ns, not significant; *** p < 0.001. The data underlying this Figure can be found in . Representative gating strategies are depicted in and raw FSC files can be found in the Figshare Data repository ( https://doi.org/10.6084/m9.figshare.c.8239897 ).

Article Snippet: For inhibitor experiments, cells were treated 24-hours post-infection with either 10 nM ATM inhibitor (Fisher, #AZD1390), 10 μM ATR inhibitor (Fisher, #NU6027), 3 mM caffeine, 16 μM DNA-PK inhibitor #1 (MedChemExpress, #NU7026), or 16 μM DNA-PK inhibitor #2 (MedChemExpress #NU7441) for 24 hours.

Techniques: Immunofluorescence, Microscopy, Translocation Assay, Residue, Infection, Control, Inhibition, Recombinant, Activation Assay, Transfection, Construct, Expressing, Flow Cytometry

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: DNA damaging agents boost the transcription of endothelin A receptor in high-grade serous ovarian cancer

doi: 10.1186/s13046-025-03607-0

Figure Lengend Snippet: Olaparib- or cisplatin-induced activation of the DNA Damage Response cascade mediates ET A R upregulation. A IB of pATM, ATM, pATR, ATR and ET A R protein expression levels of PD HG-SOC cells treated or not with 10 µM olaparib for the indicated times. Tubulin was used as loading control. B IB of pATM, ATM, pATR, ATR, pCHK1, CHK1 and ET A R protein expression levels of PD HG-SOC cells treated or not with 10 µM olaparib for 48 h. C IB of ET A R, pCHK2, CHK2 and γH2A.X protein expression levels of PD HG-SOC cells treated with ATM inhibitor (ATMi, 5 µM) and/or olaparib (10 µM). Quantification of ET A R and γH2A.X protein expression normalized to tubulin, and pCHK2 normalized to total CHK2 and tubulin. All data are presented as mean ± SD expressed as fold induction ( n = 3, * p < 0,02 vs. non-treated cells; ** p < 0,008 vs. olaparib-treated cells). D IB of ET A R, pCHK1, CHK1 and γH2A.X expression levels of PD HG-SOC cells treated with ATR inhibitor (ATRi, 2,5 µM) and/or olaparib (10 µM). Quantification of ET A R, γH2A.X protein expression normalized to tubulin, and pCHK1 normalized to total CHK1 and tubulin ( n = 3, * p < 0,02 vs. non-treated cells; ** p < 0,02 vs. olaparib-treated cells). E IB of pATM, ATM, pATR, ATR, pCHK1, CHK1 and ET A R protein expression levels of PD HG-SOC cells treated or not with 10 µM cisplatin for 48 h. F IB of ET A R, pCHK2, CHK2 and γH2A.X protein expression levels of PD HG-SOC cells treated with ATMi (5 µM) and/or cisplatin (10 µM). Quantification of ET A R, γH2A.X and pCHK2 protein expression performed as in C ( n = 3, * p < 0,04 vs. non-treated cells; ** p < 0,002 vs. cisplatin-treated cells). G IB of ET A R, pCHK1, CHK1 and γH2A.X protein expression levels of PD HG-SOC cells treated with ATRi (2,5 µM) and/or cisplatin (10 µM). Quantification of ET A R, γH2A.X and pCHK1 protein expression performed as in D ( n = 3, * p < 0,05 vs. non-treated cells; ** p < 0,04 vs. cisplatin-treated cells)

Article Snippet: ATR kinase inhibitor (AZD6738, Ceralasertib, cat. no. HY-19323, MedChemExpress, USA) was added for 48 h at 2,5 μM.

Techniques: Activation Assay, Expressing, Control

Journal: bioRxiv

Article Title: ATR inhibitors synergize with mitomycin C to enhance cytotoxicity in patient-derived non-muscle invasive bladder cancer organoids

doi: 10.1101/2025.08.29.673024

Figure Lengend Snippet: ( A , B ) BTOR58 organoids were treated for 2h with 0,5 μM gemcitabine (GEM) ( A ) or 0,5 μM epirubicin (EPI) ( B ) alone or in combination with 0,4 μM berzosertib follow-up treatment for 72h. Weekly images were taken for 6 weeks after treatment and representative images at t=1, 3, and 6 weeks are shown. Cell viability assays at t=6 weeks are shown in ( C , D ). ( E , F ) UBTOR8 organoids were treated for 2h with 4 MMC, alone or in combination with 3,2 μM ceralasertib ( E ) or 25 nM tuvusertib ( F ) follow-up treatment for 72h. Representative images at t=1, 3, and 6 weeks after treatment are presented. Cell viability assays at t=6 weeks are shown in ( G , H ). Bar graphs represent mean with SD (n=3).

Article Snippet: Next, PDOs were washed three times with PBS, before BM2+ culture medium was added, and 20h later berzosertib, ceralasertib (TargetMol #T3338), or tuvusertib (TargetMol #T10406) was added for 3 days.

Techniques: