Journal: ACS Omega

Article Title: A Bifunctional Antibody Conjugate for Marking the Location of DNA Binding Proteins on DNA Fibers

doi: 10.1021/acsomega.5c07248

Figure Lengend Snippet: Y1-PEG10 distribution shortly after IR. (a) HeLa cells were exposed to 1 Gy of ionizing radiation and fixed at different times (5 min, 15 min, 30 min, and 60 min after incubation at 37 °C. The sample treated with the kinase inhibitor Torin2 was analyzed 5 min after IR exposure. Permeabilized nuclei were incubated with an antibody against γ-H2AX and then with fluorescent secondary antibody. Blue: DAPI staining, White: γ-H2AX foci. (b) Frequency of γ-H2AX foci at different times after IR. Data represent the average of 2 independent experiments, with standard deviations shown as error bars. (c) Y1-PEG10 signals on DNA fibers from cells with or without ATR, ATM, and Torin2 kinase inhibitors harvested 5 min after exposure to 1 Gy of ionizing radiation. (d) The frequencies of Y1-PEG10 internal and terminal signals per Mb of fibers. Two independent experiments were performed. The combined total Mb of DNA analyzed from the two experiments: Control-186; AZ0156-141; VE821- 230. The total number of Y1-PEG10 signals from each condition is shown below each condition.

Article Snippet: For inhibitor studies, cells were treated for 2 h prior to, during, and following exposure to ionizing radiation either with ATM inhibitor AZD0156 (50 nM), the multikinase inhibitor Torin2 (100 μM) or the ATR inhibitor VE821 (500 nM) (all from Selleckchem).

Techniques: Incubation, Staining, Control