atm inhibitor  (MedChemExpress)

Journal: bioRxiv

Article Title: E2 displacement of CIP2A from TOPBP1 activates the DNA damage response during papillomavirus life cycles

doi: 10.1101/2025.10.15.682653

Figure Lengend Snippet: E2 induces an “ATM up ATR down” phenotype in differentiating epithelium. A. Proteins were extracted from N/Tert-1 cells with an empty vector control (N/Tert-1+Vec), expressing wild type E2 (N/Tert-1+E2-WT) or expressing an E2 that fails to interact with TOPBP1 (N/Tert-1+E2-S23A) that were growing (G), plated for differentiated with no calcium added (Day 0) or were induced to differentiate for 3 days following calcium treatment (Day 3). Western blotting with the indicated antibodies is shown. B. H&E staining of the indicated organotypic rafts. Thickness was quantitated from two independent rafts and the results shown below. C. Proteins were extracted from organotypic raft tissue generated with N/Tert-1+Vec cells, human foreskin keratinocytes immortalized by the entire HPV16 genome (HFK+HPV16) or human foreskin keratinocytes immortalized by the HPV16 E6 and E7 oncogenes (HFK+E6/E7). Western blotting with the indicated antibodies is shown. D. Proteins were extracted from W12 cells with episomal (W12 episomal) or integrated (W12 integrated) viral genomes just prior to differentiation induction (Day 0) and three days following calcium treatment (Day 3). Western blotting with the indicated antibodies is shown. E. Cells were stained with the indicated antibodies before and after 3 days of calcium induced differentiation. F. The results in were scanned and quantitated for the number of cells that had positive staining for E2 or γH2AX. G. Proximity ligation assays (PLA) were carried out for γH2AX-γH2AX interaction and for E2-γH2AX interaction before and after 3 days of calcium induced differentiation. H. The results in 1G were quantitated. * Indicates significant differences between samples, p-value<0.05. ** Indicates significant differences between samples, p-value<0.01.

Article Snippet: To inhibit ATM or ATR kinase activity, cells were treated with the ATM inhibitor, AZD0156 (MedChemExpress) or the ATR inhibitor, AZD6738 (MedChemExpress), dissolved in DMSO.

Techniques: Plasmid Preparation, Control, Expressing, Western Blot, Staining, Generated, Ligation

Journal: bioRxiv

Article Title: E2 displacement of CIP2A from TOPBP1 activates the DNA damage response during papillomavirus life cycles

doi: 10.1101/2025.10.15.682653

Figure Lengend Snippet: CIP2A and DBC1 are required for E2 activation of the DDR. A. Growing N/Tert-1+Vec (top panels), HFK+HPV16 (middle panels) and HFK+E6/E7 were stained with DAPI, B56α and CIP2A antibodies with a merge shown on the right panels. B. N/Tert-1+Vec (top panels), HFK+HPV16 (middle panels) and HFK+E6/E7 cells were differentiated by calcium treatment and three days later fixed and stained with DAPI, B56α and CIP2A antibodies with a merge shown on the right panels. C. and are representative samples that were scanned and quantitated for the levels of nuclear and cytoplasmic B56α and CIP2A and the results are presented graphically. There is a statistically significant reduction in nuclear B56α and CIP2A following differentiation of HFK+HPV16 cells as indicated with *. D. siRNA knockdown of CIP2A prevents the “ATM up ATR down” phenotype in HFK+HPV16 cells following differentiation, and the increase in E2 protein expression. E. siRNA knockdown of CIP2A increases SIRT1 levels in HFK+HPV16 cells following differentiation but does not affect DBC1 or B56α expression and prevents the increase in cyclin B1 and TOPBP1. F. siRNA knockdown of CIP2A prevents the “ATM up ATR down” phenotype in N/Tert-1+E2-WT cells following differentiation, and the increase in E2 protein expression. G. siRNA knockdown of CIP2A increases SIRT1 levels in HFK+HPV16 cells following differentiation but does not affect DBC1 or B56α expression and prevents the increase in cyclin B1 and TOPBP1. H. siRNA knockdown of DBC1 prevents the “ATM up ATR down” phenotype in HFK+HPV16 cells following differentiation, and the increase in E2 protein expression. I. siRNA knockdown of DBC1 increases SIRT1 levels in HFK+HPV16 cells following differentiation but does not affect CIP2A or B56α expression and prevents the increase in cyclin B1 and TOPBP1. J. siRNA knockdown of DBC1 prevents the “ATM up ATR down” phenotype in N/Tert-1+E2-WT cells following differentiation, and the increase in E2 protein expression. K. siRNA knockdown of DBC1 increases SIRT1 levels in HFK+HPV16 cells following differentiation but does not affect CIP2A or B56α expression and prevents the increase in cyclin B1 and TOPBP1. L. TV exonuclease assays demonstrate that CIP2A and DBC1 knockdown promotes viral genome integration irrespective of the differentiation status of the cells. Each panel pair (D-E, F-G, H-I, J-K) in was generated from the same blot, stripped and re-probed for different targets. E2 and GAPDH controls are re-used within each pair.

Article Snippet: To inhibit ATM or ATR kinase activity, cells were treated with the ATM inhibitor, AZD0156 (MedChemExpress) or the ATR inhibitor, AZD6738 (MedChemExpress), dissolved in DMSO.

Techniques: Activation Assay, Staining, Knockdown, Expressing, Generated

Journal: bioRxiv

Article Title: E2 displacement of CIP2A from TOPBP1 activates the DNA damage response during papillomavirus life cycles

doi: 10.1101/2025.10.15.682653

Figure Lengend Snippet: E2 promotes TOPBP1-ATM complex formation during differentiation and prevents TOPBP1 interaction with ATR. A. Immunoprecipitation with a TOPBP1 antibody in the presence of wild type E2 demonstrates a preferential formation of a TOPBP1-ATM complex following differentiation. The top panels show the results in N/Tert-1 cell lines, the middle panels in the HFK cell lines, and the bottom panels the W12 cell lines. B. Immunoprecipitation with an E2 antibody demonstrates that, following differentiation, wild type E2 complexes with ATM and TOPBP1 but not ATR. This phenotype depends upon E2 interaction with TOPBP1 as E2-S23A cannot interact with ATM or ATR. The top panels show the results in N/Tert-1 cell lines, the middle panels in the HFK cell lines, and the bottom panels the W12 cell lines. The input protein levels for these experiments are shown in . C. The N/Tert-1 cell lines were treated with the ATM inhibitor AZD0156 (ATMi), top two panels, or the ATR inhibitor AZD6738 (ATRi), bottom two panels. Cells were treated with inhibitor for 48 hours and then seeded for colony formation. Colonies were fixed and stained with crystal violet after 10-14 days. D. The HFK cell lines were treated with the ATM inhibitor AZD0156 (ATMi), top two panels, or the ATR inhibitor AZD6738 (ATRi), bottom two panels. Cells were treated with inhibitor for 48 hours and then seeded for colony formation. Colonies were fixed and stained with crystal violet after 10-14 days.

Article Snippet: To inhibit ATM or ATR kinase activity, cells were treated with the ATM inhibitor, AZD0156 (MedChemExpress) or the ATR inhibitor, AZD6738 (MedChemExpress), dissolved in DMSO.

Techniques: Immunoprecipitation, Staining

Journal: bioRxiv

Article Title: E2 displacement of CIP2A from TOPBP1 activates the DNA damage response during papillomavirus life cycles

doi: 10.1101/2025.10.15.682653

Figure Lengend Snippet: The “ATM up ATR down” phenotype persists in an HPV16 positive head and neck cancer cell line. A and B. Proteins were extracted from UMSCC-104 and UMSCC-47 cell lines and western blotting used to determine expression of the indicated proteins. C. The protein extracts shown in B were immunoprecipitated with TOPBP1 (top panels) or CIP2A (lower panels) and western blotted for the proteins shown. D. The protein extracts prepared in B were immunoprecipitated with SIRT1 (top panels) or DBC1 (lower panels) and western blotted for the proteins shown. E. The protein extracts prepared in B were immunoprecipitated with an acetyl lysine specific antibody and western blotted for the proteins shown. F. Immunoprecipitation with a TOPBP1 antibody demonstrates a preferential formation of a TOPBP1-ATM complex in UMSCC-104 cells and a failure of TOPBP1 to interact with ATR. This is not observed in UMSCC-47 cells that do not express E2. G. The UMSCC-104 and UMSCC-47 cell lines were treated with the ATM inhibitor AZD0156 (ATMi) for 5 days and then seeded for colony formation. Colonies were fixed and stained with crystal violet after 14-days.

Article Snippet: To inhibit ATM or ATR kinase activity, cells were treated with the ATM inhibitor, AZD0156 (MedChemExpress) or the ATR inhibitor, AZD6738 (MedChemExpress), dissolved in DMSO.

Techniques: Western Blot, Expressing, Immunoprecipitation, Staining