Journal: Science signaling

Article Title: Dynamics of p53 in response to DNA damage vary across cell lines and are shaped by efficiency of DNA repair and activity of the kinase ATM

doi: 10.1126/scisignal.aah6671

Figure Lengend Snippet: (A) Twelve cell lines were stained for p53 before or 2 hours after treatment with the DNA damaging agent NCS (100 ng/ml). Scale bar, 50μm. “M” marks the 5 melanoma lines. (B) Histograms of single cell straining intensity of p53 before (blue) or after (red) treatment with NCS. Black lines indicate the medians of the distributions. Cells are ordered by their median p53 abundance after NCS. (C and D) A549 cells were stained for p53 2 (C) or 8 (D) hours after a log2 titrations series of NCS doses. Red lines indicate the median of the distribution. The abundance of p53 did not vary significantly across DNA damage doses at 2hrs (pvalue >0.05), but did after 8hrs (pvalue <0.05, unpaired ttest). (E and F) Each cell line was stained for p53 2 (E) or 8 (F) hours after varying doses of NCS. Heatmaps represent p53 abundance. Each cell line was internally normalized (Norm.) between 0 and 1. Data in (B–F) are from N>500 cells for each cell line, representative of 2 independent experiments. AU, arbitrary units.

Article Snippet: X-ray induced DNA damage was generated with a RS-2000 source (RadSource).

Techniques: Staining

Journal: Science signaling

Article Title: Dynamics of p53 in response to DNA damage vary across cell lines and are shaped by efficiency of DNA repair and activity of the kinase ATM

doi: 10.1126/scisignal.aah6671

Figure Lengend Snippet: (A) A549 cells expressing p53-YFP were pre-treated with DMSO (NT) or Olaparib (PARPi; 10uM), NU7026 (DNA-PKi; 10uM), or CH99021 (GSK3βi; 10uM) for 1hr and then treated with NCS (100 ng/ml). Red lines represent the treated condition (thin lines, single cells; thick line, average), and the blue line represents the control (NT) cells (N>40 cells, representative of 2 independent experiments.). (B) Quantification of γH2AX intensity induced by NCS (100ng/ml; DNA damage) was measured in A549 cells after 1hr pretreatment with DMSO (NT) or the indicated inhibitor (10uM) 6 hours after treatment with 10Gy IR. Scale bar, 25μm. Histograms are shown, with a red line indicating the median (N>500 cells, representative 3 independent experiments.). (C) Images of cells from the indicated lines stained for γH2AX before or 0.5 or 24 hours after NCS treatment (100ng/ml). (D) Histograms of MCF7, A549 and HCT116 cells stained for γH2AX at the indicated time-points after NCS treatment (N > 200 cells from 2 independent experiments.). (E) γH2AX in HCT116 cells was quantified 30 min and 8 hours after NCS treatment. (F) DNA damage assessed as γH2AX abundance at 30 min and 8 hours in the indicated cell lines were compared across a range of NCS concentrations (noted in the color scale). Data are means ± S.D. (N=7 doses).

Article Snippet: X-ray induced DNA damage was generated with a RS-2000 source (RadSource).

Techniques: Expressing, Control, Staining