Deletion of the C-terminus of ATR leads to DNA damage and an increase of aberrant cells.
<p>(A - B) Western analysis of whole cell extracts from mycATR and mycATRΔC-/- cells. Cells were left untreated (NT) or treated with 0.5 mM of Hydroxyurea (HU) for 20 hours (Chronic) or treated with 5 mM of Hydroxyurea (HU) for 5 hours (Acute), washed and resuspended in fresh media. The sample...
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2025
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| Gaia: | <p>(A - B) Western analysis of whole cell extracts from mycATR and mycATRΔC-/- cells. Cells were left untreated (NT) or treated with 0.5 mM of Hydroxyurea (HU) for 20 hours (Chronic) or treated with 5 mM of Hydroxyurea (HU) for 5 hours (Acute), washed and resuspended in fresh media. The samples were collected at 0 and 5 hours after chronic treatment, and 0 and 4 hours after acute treatment. Blots were probed with α-γH2A antiserum; α-EIF1α was used as a loading control (predicted protein sizes are indicated, kDa). (C – D) Quantification of signal from the western blot showed in (A and B) using ImageJ software; statistical analysis was conducted using Graph Prism (Error bars ± SD; n = 3 experiments, * p < 0.05; ** p < 0.005; *** p < 0.001; **** p < 0.0001. Unpaired t-test). (E) Representative images acquired by a LSM880 Zeiss confocal microscope from Z stack images of mycATRΔC-/- (cl8) cells after release from chronic treatment (5 hours post treatment). Cells were fixed with 3% formaldehyde, genomic DNA stained with DAPI (cyan) and nucleus (n), kinetoplast (k) and micronucleus (n*) are indicated; α-tubulin was used to stain the cytoskeleton (gray). Bar = 12 um. (F) Percentage of populations with the indicted proportions of Nucleus/Kinetoplast (n/k) in mycATR and mycATRΔC-/- cells untreated (NT) or after release from chronic (5 hours post treatment) or acute (4 hours post treatment) HU treatment. The populations were coloured according to the proportion of n/k as normal (1n1k, 1n2n, 2n2k) or aberrant (0n1k, 1n0k, 2n1k, the presence of a micronucleus, 1M and Others for low aberrant cells as showed 0K2N). Error bars ± SD, n = 2 (>120 cells counted/experiment).</p> |
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