Figure 4. The cell cycle arrest of His^C mutant cells does not depend on the ATM/Chk2 DNA damage checkpoint.

(A and B) Wild type and His^C mutant cells responded to ionizing irradiation (IR) by phosphorylation of the variant histone H2Av, detected by a phosphospecific antibody against H2Av (γH2Av). (C and D) Without irradiation, His^C mutant cells did not show elevated γH2Av staining compared to wild type, indicating that mutant cells did not accumulate DNA damage. (E) Western blot for γH2Av using untreated (w/o HU) and HU-treated (HU) S2R+ cells as controls (two dilutions, 1 and 0.5) as well as His^C mutant embryos and wild type embryos at 5.5–6.5 hr AEL. α-Tubulin (α-Tub) was used as a loading control. HU treatment results in a significant increase of γH2Av, which was not observed in His^C mutant embryos. (F–H) BrdU pulse labelling for 15 min and staining with antibodies against BrdU and Cyclin B. lok, His^C double mutant embryos showed a similar phenotype as His^C mutant embryos (see Figure 1F). Dorsal up in (F–H), scale bars: 10 µm.

DOI: http://dx.doi.org/10.7554/eLife.02443.010

Figure 4—figure supplement 1. His^C mutant embryos show a moderate increase of γH2Av during S₁₅ progression.

His^C mutant (A–E) and wild type sibling (F–J) embryos stained with antibodies against phosphorylated H2Av (γH2Av), against β-Galactosidase (lacZ) and with DAPI to detect DNA. (A–C) His^C mutant embryos were identified by the lack of lacZ staining. (D and E) Late S₁₅ cells in the dorsal epidermis of His^C mutant embryos show a moderate increase of γH2Av as compared to ventral cells that are in early S₁₅ or still in M₁₄ (blue arrowhead). (F–H) Wild type sibling embryos of comparable age were identified based on lacZ staining. (I and J) In these embryos dorsal epidermal cells were already in S/G2₁₆. The increase in γH2Av staining was less pronounced than in His^C mutant embryos. (K–P) Untreated (w/o HU; K–M) and HU-treated (N–P) S2R+ cells were stained for γH2Av and DNA showing an increase of γH2Av signal in HU-treated cells. Dorsal up in (A–J), anterior to the left in (A–C, F–H), scale bars: 100 µm (A–C, F–H), 10 µm (D, E, I, J), 20 µm (K and N) and 5 µm (L, M, O, P).

DOI: http://dx.doi.org/10.7554/eLife.02443.011

Figure 4—figure supplement 2. His^C mutant embryos do not show accumulation of DNA damage during early embryogenesis.

(A–H) TUNEL assays with whole mount embryos to detect free 3′OH groups of DNA, which indicate DNA damage. (A–C) Wild type embryos at 4.5–5 hr AEL contained a few apoptotic cells (arrowheads). In most nuclei, free 3′OH groups were not detected. (D) Whole mount wild type embryos at 9.5–10 hr AEL showed a few apoptotic cells. (E–G) His^C mutant embryos at 6.5–7 hr AEL also contained a few apoptotic cells (arrowheads). However, the level of TUNEL staining was not uniformly elevated in the mutant cells, indicating that there is no increase of DNA damage compared to wild type. (H) His^C mutant embryos die around 9.5–10 hr AEL and show an increased number of apoptotic cells. Scale bars: 10 µm (A–C), (E–G), 100 µm (D and H).

DOI: http://dx.doi.org/10.7554/eLife.02443.012