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. 2020 Jun 4;21(8):e48920. doi: 10.15252/embr.201948920

Figure 1. CDC7 inhibition suppresses histone H2AX and RPA2 phosphorylation and DNA double‐strand break formation upon fork stalling.

Figure 1

  1. U2OS cells were either mock‐treated or treated with 10 μM XL413 for 30 min, at which point 4 mM HU was added and cells further incubated for the indicated times. Whole‐cell extracts were then analysed by Western blotting with the indicated antibodies. Reduction of phosphorylation of Ser40/41 on MCM2 is indicative of CDC7 inhibition by XL413. Data are representative of two independent experiments.
  2. U2OS cells were either mock‐treated or treated with 10 μM XL413, 4 mM HU or both for 24 h before performing neutral comet assays. Representative images of comets are shown. Scale bar = 100 μm. In the dot plots, ˜800 comets per each condition were analysed, means are indicated with a red line, and their values are shown above the plot. Data are from two independent experiments.
  3. U2OS cells were either untreated or treated with 10 μM XL413 or with 5 μM AZD6738 alone or in combination and, where indicated, after 30 min, 4 mM HU was added for a further 2 h. Whole‐cell extracts were analysed by Western blotting with the indicated antibodies. Total protein stain (TPS) is as a loading control. Data are representative of two independent experiments.
  4. U2OS cells were either untreated or treated with 4 mM HU for 5 h in the presence or absence of 10 μM XL413, which had been added to cells either 30 min prior to (pre‐treatment) or during (cotreatment) the addition of HU. Whole‐cell extracts were analysed by Western blotting with the indicated antibodies, and total protein stain (TPS) is displayed as a loading control. Data are representative of two independent experiments.

Source data are available online for this figure.