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1.
Figure 7

Figure 7. From: Chk1 is an essential kinase that is regulated by Atr and required for the G2/M DNA damage checkpoint.

A model for the mammalian DNA damage response pathway (See text for details). The question marks indicate hypothetical regulatory interactions.

Qinghua Liu, et al. Genes Dev. 2000 June 15;14(12):1448-1459.
2.
Figure 5

Figure 5. From: Chk1 is an essential kinase that is regulated by Atr and required for the G2/M DNA damage checkpoint.

Kaplan-Meier plot of tumor incidence in CHK1+/+ and CHK1+/− WNT-1 transgenic females. Twenty-two animals of each genotype were monitored for mammary tumor formation for ∼10 months. Tumor-free survival is plotted against the animal age in days.

Qinghua Liu, et al. Genes Dev. 2000 June 15;14(12):1448-1459.
3.
Figure 6

Figure 6. From: Chk1 is an essential kinase that is regulated by Atr and required for the G2/M DNA damage checkpoint.

Chk1 is phosphorylated on S345 after DNA damage and this is regulated by Atr. (A) Phosphorylation of Chk1 on S345 after DNA damage. 293T cells were untreated or treated with IR (20 Gy and harvested after 1 hr), UV (50 J/m2 and harvested after 2 hr) or HU (1 mm for 24 hr). Whole cell lysates were immunoprecipitated (IP) with rabbit anti-Chk1 or anti-p-S345 antibodies followed by immunoblotting with mouse anti-Chk1 antibodies. (B) Overexpression of wild-type (WT) but not kinase-deficient (KD) Atr enhances S345 phosphorylation of cotransfected Chk1. Thirty-six hours after cotransfection of CMV:Chk1 and CMV:FLAG-Atr–WT or CMV–FLAG–Atr–KD, 293T cells were untreated (−) or treated with UV (50 J/m2) and harvested after 1.5 hr. Whole cell lysates were immunoblotted with anti-FLAG antibodies to detect Atr expression, or assayed for S345 phosphorylation of Chk1 by IP–Western blot as described in (A). (C) Induction of kinase-deficient Atr inhibits S345 phosphorylation of endogenous Chk1. GM847/ATR–KD cells were cultured in the absence (−) or presence (+) of 1 μg/ml doxycycline (Dox) for 48 hr, untreated (−) or treated with UV (50 J/m2) and harvested after 1 (1h) or 4 (4h) hr. Whole cell lysates were immunoblotted with anti-FLAG or mouse anti-Chk1 antibodies to detect Atr–KD or endogenous Chk1 expression. And IP–Western was performed to detect S345 phosphorylation of Chk1 as described in A.

Qinghua Liu, et al. Genes Dev. 2000 June 15;14(12):1448-1459.
4.
Figure 4

Figure 4. From: Chk1 is an essential kinase that is regulated by Atr and required for the G2/M DNA damage checkpoint.

CHK1−/− cells are defective for the G2/M DNA damage checkpoint. (A) FACS (DNA content) analysis of irradiated GFP- or Cre-transfected CHK1+/+, CHK1flox/+, and CHK1flox/− cells. Twenty-four hours after transfection, cells were subjected to 10 Gy IR, harvested at 0, 12, 18, and 24 hr and stained with PI. The percentage of G1–S and G2 population (mitotic cells are included in the G2 counts but are generally a small proportion) is shown above each FACS sample. (B) FACS (DNA content) analysis of IR and nocodazole-treated cells. Cells of the indicated genotypes were transfected and irradiated as in A and incubated in nocodazole (0.2 μg/ml) containing media 30 min after irradiation. (C) Images (1000×) of the DAPI-stained 12-hr samples in B. Arrows indicate mitotic cells with condensed chromosomes and no nuclear membrane. (D) A mitotic index graph of IR and nocodazole-treated cells described in B and C. Cells (250–300) were counted for each sample. GFP or Cre transfections are indicated by open or closed circles. f5 and f6 represent two independent CHK1flox/− cell lines and f7 is a control CHK1flox/+ cell line.

Qinghua Liu, et al. Genes Dev. 2000 June 15;14(12):1448-1459.
5.
Figure 2

Figure 2. From: Chk1 is an essential kinase that is regulated by Atr and required for the G2/M DNA damage checkpoint.

In vitro culture of preimplantation embryos. (A) In vitro culture of blastocysts isolated from CHK1+/− intercrosses. A typical image at day 0 and day 3 are shown along with the percentage of each category. (TE) Trophoectoderm; (ICM) inner cell mass. All images are 320× except when otherwise indicated. (B) In vitro culture of eight-cell morula. Shown here are a litter of 10 embryos isolated from a CHK1+/− intercross. The images were captured at 6, 30, and 45 hr in culture (100× except when otherwise indicated). The asterisks indicate the empty zona pellucida after the embryos have hatched. The embryos were individually picked and labeled 1–10. Embryos 1, 2, 3, and 10 failed to hatch. (C) PCR genotyping of embryos from B. The lane numbers correspond to that of the embryos. Embryo 6 was lost during transfer and embryo 3 was too degenerate to give any products. (WT) Wild type; (MT) mutant. The asterisk indicates a nonspecific band generated by primers alone. (D) TUNEL analysis of blastocysts derived as described in B (100×). (E) Confocal images (DAPI, 1000×) of blastocysts analyzed in D. The arrows indicate condensed and fragmented nuclei.

Qinghua Liu, et al. Genes Dev. 2000 June 15;14(12):1448-1459.
6.
Figure 1

Figure 1. From: Chk1 is an essential kinase that is regulated by Atr and required for the G2/M DNA damage checkpoint.

CHK1 disruption results in early embryonic lethality. (A) Disruption of the CHK1 gene in ES cells. (a) Chk1 protein structure. Located at the amino terminus are the “G×G××G” ATP-binding motif and kinase domain. (b) Restriction map of the wild-type CHK1 locus including exons 2–7 and location of the 3′ external and 5′ internal probes used for Southern blots shown below. (c) Restriction map of the neo targeting vector. The direction of transcription is shown by arrows beneath the neo and TK markers. (pBS) pBluescript plasmid. (d) Predicted restriction map of the targeted CHK1 allele. Only relevant restriction sites are shown. (Nc) NcoI; (Nd) NdeI; (Rv) EcoRV. (B) Identification of CHK1+/− ES clones by Southern blot analysis. (a) Genomic DNA was digested with NcoI and EcoRV and probed with the 3′ external probe. Genotypes are shown above each lane. CHK1+/− ES clones showed a 10.5-kb wild-type and a 6.5-kb mutant band as predicted. (b) Genomic DNA was digested with NdeI and probed with the 5′ internal probe. CHK1+/− ES clones displayed a 9.5-kb wild-type and a 7.0-kb mutant band. (c) CHK1 genotyping by PCR. The wild-type and mutant CHK1 alleles generate a 282-bp and a 572-bp band, respectively. (C) Morphological and histological analysis of E7.5 embryos isolated from CHK1+/− intercrosses. A typical photograph is shown for normal (a,c) and mutant (b,d) E7.5 embryos freshly dissected from decidua (a,b) or hematoxylin and eosin-stained sections of E7.5 decidua (c,d).

Qinghua Liu, et al. Genes Dev. 2000 June 15;14(12):1448-1459.
7.
Figure 3

Figure 3. From: Chk1 is an essential kinase that is regulated by Atr and required for the G2/M DNA damage checkpoint.

Construction and analysis of a conditional CHK1-deficient ES cell line. (A) Restriction maps of targeting vectors and targeted alleles. (a) hprt targeted allele; (b) hprt targeting vector; (c) wild-type CHK1 locus; (d) flox targeting vector; (e) floxneo-targeted allele; (f) flox allele generated after neo excision; (g) flox Δ allele generated after excision of exon 2. Only relevant restriction sites are shown. (Nc) NcoI; (Nd) NdeI; (Rv) EcoRV. (B) A schematic representation of the construction of conditional CHK1-deficient ES cells. In brief, a CHK1 floxneo/+ cell line was first obtained using the flox-targeting vector followed by the Cre–loxP-mediated neo excision to create CHK1flox/+ cells. The remaining wild-type gene was then disrupted in the CHK1flox/+ cells by the hprt targeting vector to generate CHK1flox/− cells. Finally, CHK1flox/− cells were conditionally converted into CHK1−/− cells by excision of exon 2 by transient transfection of PGK:Cre. (C) A Southern blot showing excision of the flox allele in CHK1flox/+ and CHK1flox/− cells. Both are sister cell lines obtained from the same screen for generating CHK1flox/− cells. The asterisk refers to a band produced by random integration of the hprt construct in the CHK1flox/+ cells. Genomic DNA was prepared from untransfected (lanes 1,6) or Cre-transfected cells (lanes 2–5 and 7–10) collected at 24, 48, 72, and 96 hr after electroporation, digested with NdeI and EcoRV and probed with the 5′ internal probe. Δ refers to the excised flox allele. The faint wild-type band present in lanes 2–4 was contributed by the feeder cells. (D) Comparison of Cre-transfected CHK1flox/+ and CHK1flox/− cells by FACS (DNA content) analysis. Cells were harvested at 24, 48, and 72 hr after electroporation and stained with propidium iodide (PI). The arrow indicates cells with less than 2N DNA content that were present in the 72-hr flox/− sample.

Qinghua Liu, et al. Genes Dev. 2000 June 15;14(12):1448-1459.

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