Sidi et al., 2008 - Chk1 Suppresses a Caspase-2 Apoptotic Response to DNA Damage that Bypasses p53, Bcl-2, and Caspase-3. Cell   133(5):864-877 Full text @ Cell

Fig. 1 A Morpholino Screen Identifies chk1 as a Loss-of-Function Suppressor of p53e7/e7-Associated Radioresistance

(A) Live 25 hpf embryos of the indicated genotypes stained with AO at 7.5 hpIR (12.5 Gy). Anterior, left. Note the complete absence of AO labeling in the brain and spinal cord of the irradiated p53 mutant.

(B) MO screen for loss-of-function suppressors of p53e7/e7-associated radioresistance. Noninjected and 1 cell-stage MO-injected embryos were irradiated at 18 hpf (12.5 Gy). AO uptake by cells was quantified by analyzing images of whole embryos photographed live at 7.5 hpIR (y axis) (images as in C). Injected MOs are indicated along the x axis. Bars are color coded and refer to the genetic background used for injections (gray, p53+/+; black, p53e7/e7). AO staining was quantified in ≥8 embryos per knockdown, with 50 or more embryos scored per knockdown (except †> 1000); ‡, embryos showed developmental defects. All data are reported as means ± SEM. Statistical significance versus the noninjected p53e7/e7 response: * p < 0.05; ** p < 0.005; *** p < 0.0005; ns (not significant), (two-tailed Student's t test).

(C) Fluorescent images of AO-labeled, live p53 mutants injected with indicated MOs and representative of the phenotypes quantified in (B).

Fig. 2 chk1 Knockdown Radiosensitizes p53 Mutants but Is Otherwise Compatible with Normal Zebrafish Development

(A) Fluorescent images of representative embryos of indicated genotypes +/- chk1 MO after 0 or 12.5 Gy IR; 5bpmmMO (5 base pair mismatch MO).

(B) Quantified AO responses of indicated genotypes with or without IR (12.5 Gy) and chk1 MO. Gray bars, p53+/+ background; black bars, p53e7/e7 background. AO staining was quantified in ≥8 embryos per condition, with > 1000 embryos scored. All data are reported as means ± SEM *** p < 0.0001 (two-tailed Student's t test).

(C) Western blots comparing the levels of Chk1, Chk2, and phosphorylated Cdc2 (Tyr15) in protein lysates from 25.5 hpf embryos injected with the indicated MOs.

(D) Nonirradiated p53+/+;chk1MO larva photographed live at 5 days postfertilization (dpf) show no apparent developmental defects but is slightly delayed (smaller swim bladder). Such larvae survived to adulthood.

(E) Fluorescent images of representative irradiated embryos of indicated genotypes. p53e6 is the N168K mutation, corresponding to human residue 200. p53MO, MO against the p53 5′UTR.

(F) Fluorescent images of live transgenic embryos injected with the indicated MOs at the 1-cell stage and expressing EGFP in the notochord (top row, embryos photographed at 24 hpf) or in myeloid progenitors (bottom row, embryos photographed at 16.5 hpf). Tg(myoD:EGFP) and Tg(pu.1:EGFP) embryos were treated with or without IR (12.5 Gy) at 18 hpf and 10 hpf, respectively. Insets, higher magnification views of GFP-expressing cells. Top row, lateral views, anterior to the left. Bottom row, dorsal views, anterior facing down.

(G) Quantification of myeloid cells in 28 hpf embryos generated as indicated (x axis) and processed as in (H). Gray bars, p53+/+ background; black bars, p53e7/e7 background. mpo/l-plastin staining was quantified in ≥15 embryos per condition. Data are reported as means ± SD ** p < 0.001, *** p < 0.0001 (two-tailed Student's t test). Note that while the numbers of mpo/l-plastin-positive cells are reduced ∼3-fold in IR-treated versus untreated p53+/+ embryos; they are unchanged in treated versus untreated p53e7/e7 embryos. Also note that chk1 knockdown induces an average 2-fold reduction in myeloid cell numbers in the p53e7/e7 background after IR.

(H) Images of representative 28 hpf embryos of indicated genotypes processed for in situ hybridization of mpo and l-plastin riboprobes (blue, differentiated granulocytes and monocytes) and band 3 (red, erythrocytes). Note the specific reduction in number of granulocytes/monocytes.

Fig. 3 IR-induced p53-Independent Apoptosis after Chk1 Loss Occurs Cell Autonomously and Independently of Caspase-3

(A) Fluorescent images of 25 hpf embryos (anterior, left). TUNEL reactivity after IR (0 or 12.5 Gy) recapitulates live AO labeling (see Figure 2A).

(B) Embryos from the same experiment immunostained with an antiactivated-Caspase-3 antibody. Note the absence of immunoreactivity in the irradiated p53e7/e7;chk1MO embryo.

(C) Electron micrographs (sagittal sections) of the CNS in embryos of indicated genotypes after 0 or 12.5 Gy IR. Gö6976 is a specific Chk1 inhibitor (see Figure 5, Figure 6 and Figure 7). Lower row, 2.5x closeups on the areas boxed in yellow in upper panels. Note multiple cells with stereotypical chromatin compaction/segregation in columns 2 and 4, as opposed to healthy nuclei in columns 1 and 3. Organelles and plasma membrane are intact in the shown Chk1-inhibited irradiated p53 mutant cell, as expected from an apoptotic (as opposed to necrotic) event. See Figure S2 for more details. Scale bar, 2 μM.

(D) Experimental procedure for the generation of the genetic chimeras shown in (E).

(E) 5-μm-thick confocal sections of spinal cords in irradiated chimeras. TMR Dextran (red) marks the donor cells. TUNEL shown in green. First row, cells from a p53e7/e7 embryo that was injected with the chk1 MO at the 1-cell stage (p53e7/e7;chk1MO embryo) transplanted into a p53e7/e7 host. Second row, p53e7/e7 cells transplanted into a p53e7/e7;chk1MO host.

Fig. 4 Genetic Dissection of the Zebrafish Chk1-Suppressed Apoptotic Pathway

(A) Quantified AO labeling in spinal cords of 12.5 Gy-exposed p53e7/e7;chk1MO embryos injected with H2O (bar on the far left) or the indicated MOs (x axis). AO staining was quantified in e8 embryos per MO with a total of ≥100 embryos scored. All data are means ± SEM *** p < 0.0001 (two-tailed Student's t test).

(B) Fluorescent images of representative embryos from the experiments shown in (A).

(C) At left, RT-PCR of casp2 transcripts from embryos either injected or not injected with casp2 MO. At right, schematics of caspase-2 protein variants (top, wild-type protein; bottom, predicted protein translated from exon 4-deleted transcripts).

(D) Fluorescent images of embryos of the indicated genotypes with or without IR (12.5 Gy at 18 hpf), chk1 MO, or bcl-xl mRNA. Numbers in brackets refer to the corresponding bars in (E).

(E) Quantified AO responses (n ≥ 8) for embryos of indicated genotypes +/- bcl-xl mRNA. Gray bars, p53+/+ background; black bars, p53e7/e7;chk1MO background. Numbers in brackets refer to the representative-embryo images in (D). Data are means ± SEM.

Fig. 5 The Chk1-Suppressed Pathway Is Conserved in HeLa Cells

(A) Western blots comparing the levels of caspase-2 (pro and cleaved forms) and cleaved caspase-3 at 24 hpIR in lysates from HeLa cells carrying or not carrying a BCL2 transgene (Tg[BCL2]) and treated with or without IR (10 Gy) or Chk1 inhibitor (Gö6976, 1 μM).

(B) Analysis of HeLa cell survival at 72 hpIR (0 Gy versus 10 Gy) in the presence or absence of Gö6976 and/or BCL2. Gö6976 radiosensitizes the cells ∼2-fold regardless of the BCL2 transgene (compare bars 5 and 6, and bars 7 and 8). Note that BCL2 is functional (i.e., radioprotective) in these experiments (compare lanes 5 and 7). Data are means ± SEM.

(C) Fluorescent images of HeLa Tg(Cyt-c-GFP) cells with or without Tg(BCL2) or Gö6976 at 24 or 48 hpIR (10 Gy). Note the punctate GFP patterns in all 24 hpIR samples and the diffuse GFP pattern in the 48 hpIR sample.

(D) Levels of cleaved caspase-2 and caspase-3 at 24 hpIR (10 Gy) in HeLa cells transfected with LACZ or CHK1 siRNAs at 72 hr before IR.

(E) Western blots comparing the activities of Chk1 (Cdc2 phosphorylation at Tyr15 and CDC25C phosphorylation at Ser216) and MK-2 (Hsp-27 phosphorylation at Ser82) following exposure to IR and increasing concentrations of Gö6976.

(F) MK-2 phosphorylates Hsp-27 in HeLa cells. Western blot of lysates from irradiated HeLa cells exposed to increasing concentrations of the p38MAPK specific inhibitor SB203580 (Reinhardt et al., 2007), showing a dose-dependent reduction in phosphorylated Hsp-27.

(G) Knockdown efficiencies of the indicated shRNAs as measured by western blots with anticaspase-2 and anticaspase-3 antibodies.

(H) Effects of GFP, CASP2, and CASP3 shRNAs on apoptotic cell numbers at 48 hpIR as measured by AnnexinV (+) / PI (-) staining of HeLa cells treated with 10 Gy with or without Gö6976 (1 μM). For each shRNA, the average apoptotic cell number (given as % of GFP shRNA control) is shown. All data are means ± SD ** p < 0.01 (two-tailed Student's t test). Asterisks on top of bars refer to comparisons with GFP shRNA.

(I) Synergistic activation of ATM and ATR by Gö6976 and IR. Western blots comparing the activities of ATM (Chk2 phosphorylation at Thr68) and ATR (Chk1 phosphorylation at Ser317) after 0 or 10 Gy IR with or without Gö6976 (1 μM). Levels of DNA damage were detected with an antiphospho-H2A.X antibody.

(J) Cell-cycle distribution of HeLa cells undergoing Chk1-suppressed apoptosis. HeLa cells harboring GFP or CASP2 shRNAs and treated with or without 10 Gy IR with or without Gö6976 (1 μM), as indicated, were fixed at 48 hpIR and stained for TUNEL and PI. For each shRNA line, upper panels show PI-single histograms and lower panels show PI/TUNEL double-staining images. Cell-cycle phases and threshold for TUNEL positivity are indicated in red and green, respectively, in each no-treatment control images.

(K) Quantification of the TUNEL stains shown in (J). Data are means ± SEM * p < 0.05 (two-tailed Student's t test).

(L) Quantified data from experiment in (J) expressed as means ± SEM ** p < 0.002 (two-tailed Student's t test). White bars indicate cells dying in G1 phase. Black bars indicate cells dying in S phase. Grey bars, cells dying in G2 phase.

Fig. 6 Influence of Genetic Background on Gö6976-Mediated Radiosensitization of Human Cancer Cells

(A) Western blots comparing the levels of caspase-2 (pro and cleaved forms) and cleaved caspase-3 in 24-hpIR lysates from TP53+/+ and TP53-/- HCT116 cells that were treated with or without IR (10 Gy) or Gö6976 (1 μM).

(B) Analysis as in (A) of the SAOS2 (left), MDA-MB-435 (middle), and LN-428 (right) lines.

(C) Apoptotic cell numbers at 48 hpIR as measured by Annexin V (+) / PI (-) staining of the indicated cell lines treated with 0 or 10 Gy IR with or without Gö6976 (1 μM). Data are means ± SEM. See Figure S9 for a CHK1 shRNA-mediated phenocopy of Gö6976 in TP53-/- HCT116 cells.

Fig. 7 Effects of Gö6976 in Zebrafish In Vivo Models of p53 Loss and bcl-2 Gain

(A) Fluorescent images of AO-labeled embryos of indicated genotypes photographed at 25.5 hpf. Embryos were exposed to 0 or 12.5 Gy IR and to the indicated drugs at 18 hpf. Gö6976, specific Chk1 inhibitor (1 μM). Chk2 Inhibitor II, specific Chk2 inhibitor (10 μM); KU55933, specific ATM inhibitor (10 μM). Note the range of toxicities in nonirradiated p53+/+ embryos treated with KU55933 or Chk2 Inhibitor II, with strong AO labeling preferentially localized in the brain and eyes (first column, third and fourth rows), as opposed to the Gö6976-treated embryo (first column, second row). Inversely, note the strong IR-induced AO labeling in the Gö6976-treated p53 mutant (last column, second row), but the lack of staining in the mutants treated with KU55933 or Chk2 Inhibitor II (last column, third and fourth rows).

(B) Temporal requirement for Chk1 loss with respect to IR. p53 mutant embryos were exposed to Gö6976 for the indicated times. AO staining was quantified on a scale from “-” to “+++”with “-” representing the p53 mutant response and “+++” the response of sibling mutants treated with Gö6976 for 6 hr (∼500-fold greater response).

(C) Fluorescent images of 9 dpf zebrafish larvae carrying the indicated transgene. Larvae were treated with 0 Gy or 15 Gy IR at 5 dpf and were exposed to Gö6976 (or DMSO as control) for a total of 5 days starting at 4 dpf. White arrowhead indicates the position of the thymus. Note the absence of detectable GFP in the Gö6976-treated Tg(rag2:EGFP-bcl-2) irradiated larva.

(D) Simplified model for the vertebrate apoptotic response to DNA damage, highlighting the p53-independent pathway normally blocked by IR-activated Chk1 (CS, for Chk1-suppressed pathway), which is distinct from the classical intrinsic (mitochondrial, MIT) and extrinsic (death-receptor, DR) pathways. See text for details.

Fig. S1 . Chk1 depletion eliminates the IR-induced G2/M checkpoint in zebrafish
(A) Cell-cycle profiles of zebrafish embryos of the indicated genotypes (6 hpIR) as determined by flow cytometry. DNA content analyzed by PI staining. Note that cells from irradiated chk1 morphants fail to accumulate in G2 at 6 hpIR, regardless of p53 status.
(B) 4N DNA ratios (6 hpIR / 0 hpIR) as determined by flow cytometry of PI-stained whole embryo homogenates. Embryos were irradiated (12.5 Gy) at 18 hpf. Data collected from 3 independent experiments are reported as means ± SD.
(C) Fluorescence images of representative embryos of indicated genotypes immunostained with an anti-phospho histone H3 antibody. Note that chk1MO embryos analyzed at 2 hpIR show dramatically increased numbers of phospho-histone H3 (pH3)-positive (mitotic) cells compared with chk1WT embryos, again irrespective of their p53 genotype.
(D) Quantification of the pH3 immunostainings shown in panel C. Percentages of pH3 positive cells are means ± SEM. ** P<0.01 (two-tailed Student’s t-test). Note that the mitotic phenotype of chk1MO embryos was only transient, consistent with a checkpoint defect.

Fig. S2 Cytologic hallmarks of apoptosis, but retention of elevated procaspase-3 levels, in irradiated Chk1-inhibited p53e7/e7 embryos
Electron micrographs (sagittal sections) of wild-type versus Chk1-inhibited p53 mutant CNS after 12.5 Gy IR. Gö6976 is a specific Chk1 inhibitor (see Figures 5-7). Cytologic hallmarks of apoptosis (as defined in Wyllie et al., 1980) are shown as follows.
(A) Nuclear chromatin compaction and segregation alongside retention of intact cytoplasmic organelles and plasma membrane (white arrowheads and dashed yellow outline, respectively, in panel A′).
(B) Nuclear chromatin compaction and segregation alongside cytoplasmic condensation. In B′, the plasma membrane is outlined in yellow and the nuclei of surrounding healthy cells are indicated by white asterisks. Compare the size of the apoptotic cell to the size of healthy nuclei.
(C-E) Nuclear morphology of early (C), mid-stage (D) and late stage (E) apoptosis.
(C) Nuclear chromatin compaction and segregation.
(D) Nuclear budding.
(E) Nuclear fragmentation.
Scale bar, 1 μM.
(F) Western blot comparing the levels of procaspase-3 in wild-type versus p53 mutant embryos 7.5 hr after 0 or 12.5 Gy IR in the presence or absence of the Gö6976. Note that IR leads to a significant reduction in procaspase-3 levels in wild-type embryos exposed or not exposed to the inhibitor, as expected from cleavage of the pro-form. In contrast, no such decrease in procaspase-3 levels is observed in isogenic irradiated p53 mutants even after exposure to the inhibitor (lane 8), even though Gö6976 restored IR-induced cell death with complete penetrance in these mutants (Figure 7A). The anti-caspase-3 antibody used in this experiment is the rabbit anti-human caspase-3 pAb from Stressgen (AAS-103) that recognizes procaspase-3 in all species thus far tested, including Xenopus. The band showing reduction in irradiated wild-type embryos migrates between the 25 and 37 kDa markers, consistent with the predicted sizes of zebrafish procaspase-3a and procaspase-3b (31 kDa), strongly supporting cross-reactivity.

Fig. S3 Validation of the cell transplantation assay
See Figure 3D for details on experimental procedure (cell transplantation and IR exposure).
(A) Dorsal view of a 5 μm thick confocal section of a p53+/+ spinal cord. TMR Dextran (red) marks cells from a p53+/+ donor embryo, which were transplanted at the blastula stage. TUNEL shown in green. The genetic chimera was not irradiated. Note that the transplanted p53+/+ cells do not stain TUNEL positive, showing that the transplantation technique does not induce apoptosis.
(B,C) 5 μm thick confocal sections of spinal cords in irradiated chimeras. TMR Dextran (red) marks the donor cells. TUNEL shown in green. (B), p53+/+ cells transplanted into a p53e7/e7 host. (C), p53e7/e7 cells transplanted into a p53+/+ host. Consistent with the fact that wild-type zebrafish embryos respond to IR through the cell autonomously-acting, mitochondrial apoptotic pathway (Berghmans et al., 2005; Kratz et al., 2006), p53+/+ cells transplanted into p53e7/e7 hosts stained TUNEL-positive after IR (86%, n=108) while neighboring p53e7/e7 cells remained largely TUNEL-negative (panel B). Furthermore, 86% (n=73) of p53e7/e7 cells transplanted into p53+/+ hosts remained refractory to IR-induced death, as indicated by their TUNEL negativity within an otherwise TUNEL-positive environment (panel C). Hence, TUNEL reactivity of transplanted cells after IR strictly depends on the p53 genotype of a cell, occurs irrespective of the cellular environment, and has very little, if any, influence on neighboring cells.

Fig. S4 Chk1-suppressed apoptosis in both zebrafish embryos and human cancer cells is largely Chk2-independent
(A) AO uptake in embryos injected at the 1-cell stage with indicated MOs (x axis) was quantified by analyzing images of whole embryos photographed live at 7.5 hpIR (12.5 Gy) (images as in Figure 1A, C). Bars are color-coded and refer to the genetic background used for injections (gray, p53+/+; black, p53e7/e7). AO staining was quantified in ≥8 embryos per knockdown, with 50 or more embryos scored per knockdown (except †, >1000). Data are reported as means ± SEM. * P<0.05; ** P<0.005; *** P<0.0005 (two-tailed Student’s t-test). Note the modest (∼30%) reduction in AO reactivity in p53e7/e7;chk1MO;chk2MO embryos compared to p53e7/e7;chk1MO embryos.
(B) Western blot showing the levels of pro- and cleaved caspase-2 at 24 hpIR (10 Gy) in HeLa cells that were treated or not treated with Gö6976 for 24 hr, and that were transfected at 72 hr prior to treatment with siRNAs against LACZ or CHK2. Whereas the CHK2 siRNA strongly knocked down Chk2, it failed to block caspase-2 cleavage.
(C) Analysis of HeLa cell survival at 72 hpIR (0 Gy vs 10 Gy) in the presence or absence of G&oum;6976 (1 μM) and in the presence of a LACZ or CHK2 siRNA (black and white bars, respectively). Whereas CHK2 siRNA confers protection to IR alone, it fails to protect cells treated with both IR and Gö6976.

Fig. S5 Knockdown efficiencies of selected MOs
(A) Western blot of 25.5 hpf zebrafish protein lysates from non-injected and atr MO-injected embryos using an anti-human ATR antibody recognizing an internal peptide that is highly conserved in zebrafish Atr. Detection of a high molecular weight zebrafish band running similarly to human ATR (lane 1) supports crossreactivity of the antibody with zebrafish ATR (the predicted molecular weight of zebrafish ATR, 300 kDa, matches that of human ATR). Relative band intensities show that the specific atr MO (Stern et al., 2005) knocks downs zebrafish ATR levels in a dose dependent fashion. The MO concentration used in the MO screen (Figure 1) and epistasis analyses (Figure 4), 0.25 mM, leads to a >50% reduction in ATR levels compared to control (compare lanes 5 with 2 and 3).
(B-E) Gel migration profiles of RT-PCR products from non-injected and MO-injected embryos using primers in exons flanking the targeted exon (Table S1; primers sequences are available upon request). All bands were excised and sequenced. Predicted protein products are indicated at the right. All RT-PCRs are semi-quantitative with a β-actin amplicon serving as loading control.
(B) atm. Left primer is located in exon 54, right primer is located in exon 58 (Imamura and Kishi, 2005). The atm MO interferes with splicing at the intron 55/exon 56 splice junction, resulting in either retention of intron 55, deletion of exon 56, or both. Both aberrant splice products result in early stop codons (as a result of in-frame reading of the intron or of a frameshift caused by exon skipping), which is predicted to result in an early truncation of the ATM protein before the PIKK kinase domain. Numbers below the lanes refer to embryo morphology at 18 hpf. 1-3, 5: normal; 4, grossly affected or dead, incompatible with the IR/AO assay. Note that the atm MO concentration used in our study, which is compatible with our IR/AO assay, leads to an incomplete knockdown of atm. As exemplified in lane 4, this same MO concentration could lead to stronger knockdowns, but in this case the embryos could not be scored in the assay.
(C) p63. Left primer is located in exon 2, right primer is located in exon 4. The p63 MO interferes with splicing at the exon3/intron 3 splice junction, resulting in retention of intron 3. This aberrant splice product results in an early stop codon (as a result of in-frame reading of the intron) predicted to result in an early truncation of any p63 protein expressed from the p63 locus before the DNA binding domain (which is essential for the activities of both pro- and anti-apoptotic isoforms of p63). p63 MO strongly depletes the wild-type p63 mRNA pool, with the majority of transcripts retaining intron 3, leading to an efficient gene knockdown.
(D) casp8. Left primer is located in exon 2, right primer is located in exon 4. The casp8 MO interferes with splicing at the exon3/intron 3 splice junction, resulting in retention of intron 3. This aberrant splice product results in an early stop codon (as a result of in-frame reading of the intron), predicted to result in an early truncation of procaspase-8, thus removing part of the second DED domain and the entirety of the catalytic domain. casp8 MO both strongly depletes the wild-type casp8 mRNA pool and attenuates the levels of both wild-type and aberrant splice forms, resulting in a highly efficient gene knockdown.
(E) casp9. Left primer is located in exon 1, right primer is located in exon 3. The casp9 MO interferes with splicing at the exon2/intron 2 splice junction, resulting in retention of intron 2. This aberrant splice product results in an early stop codon (as a result of in-frame reading of the intron), predicted to result in an early truncation of procaspase-9, thus removing part of the CARD domain and the entirety of the catalytic domain. casp9 MO results in an incomplete, ∼50% knockdown, but higher concentrations of the MO were either lethal prior to 18 hpf, or viable but extremely toxic to the embryos, precluding the analysis of epistatic relationships with chk1 and p53.
(E) Gel migration profiles of RT-PCR products from non-injected and casp2 MO2-injected embryos using primers in exons flanking exon 3 (left primer is located in exon 2, right primer is located in exon 4). Predicted protein products are indicated at the right.
(F) Quantified AO responses of irradiated (12.5 Gy) p53e7/e7;chk1MO embryos that were non-injected (left) or injected with casp2 MO2 (right). AO staining was quantified in the spinal cords of ≥9 embryos per condition, with ≥50 embryos scored. The data are reported as means ± SEM. Statistical significance estimated via a two-tailed Student’s t-test.

EXPRESSION / LABELING:
Gene:
Antibody:
Fish:
Knockdown Reagent:
Anatomical Term:
Stage: Prim-5

Fig. S6 puma depletion abrogates IR-induced apoptosis in wild-type zebrafish embryos
Lateral views of 25.5-hpf wild-type zebrafish embryos that were injected at the 1-cell stage with the indicated MOs, irradiated (0- or 12.5-Gy) at 18 hpf, and immunostained with an anti-active-caspase-3 antibody. The p53 MO-injected embryo serves as an internal control. Note that puma MO, but not puma 5bpmmMO, produces a complete phenocopy of p53 MO.

EXPRESSION / LABELING:
Antibody:
Fish:
Condition:
Knockdown Reagents:
Anatomical Term:
Stage: Prim-5

Fig. S7 Chk1-suppressed apoptosis occurs predominantly during the interphase in zebrafish embryos
(A) Confocal images of the caudal spinal cord in representative embryos of each indicated genotype processed for TUNEL (green)/pH3 (red) double-labeling. While approximately half of the pH3-labeled cells in irradiated p53e7/e7;chk1MO embryos are TUNEL positive (see panel B), the bulk of TUNEL-stained nuclei are pH3 negative, implying that death mainly occurs during the interphase. By contrast, cisplatin- or doxorubicin-induced death of MK-2–depleted Tp53-/- MEFs occurs exclusively in mitosis (Reinhardt et al., 2007).
(B) Percentage of double-labeled cells among >200 pH3-positive cells visualized on images as in panel A. Data are means ± SEM.

Fig. S9 CHK1 shRNAs phenocopy Gö6976 in HCT116 cells
(A) Apoptotic cell numbers at 48 hpIR as measured by Annexin V (+) / PI (-) staining of cell lines expressing the indicated shRNAs. Cells were treated with 0 or 10 Gy IR (white and black bars, respectively). All data are reported as means ± SEM. Asterisks on top of bars refer to comparisons with GFP shRNA + IR. * P<0.05; ** P<0.01; *** P<0.005 (two-tailed Student’s t-test).
(B) Western blot comparing the levels of Chk1 in 24-hpIR lysates from the experiment shown in panel A.

Fig. S10 Extreme IR-induced DNA damage fails to force apoptosis in zebrafish p53 mutants endowed with wild-type Chk1 activity
(A) Western blot comparing the levels of phosphorylated H2A.X in protein lysates from p53 mutant embryos 0.5 hr after 0, 12.5, 75 or 150 Gy IR in the presence or absence of the specific Chk1 inhibitor Gö6976 (1 μM). Acridine orange (AO) reactivity at 7.5 hpIR of embryos from the same experiment is indicated below the blot (see panel B for images of representative embryos). Note that AO reactivity does not correlate with levels of DNA damage. Specifically, IR doses up to 150 Gy (which lead to dramatic levels of DNA damage) are insufficient to mimic the combinatory effects of 12.5 Gy + Chk1 inhibitor treatment.
(B) Fluorescence images of AO-stained embryos from the experiment described in A. Corresponding western blot lanes are indicated in upper left corners.

Acknowledgments:
ZFIN wishes to thank the journal Cell for permission to reproduce figures from this article. Please note that this material may be protected by copyright.

Reprinted from Cell, 133(5), Sidi, S., Sanda, T., Kennedy, R.D., Hagen, A.T., Jette, C.A., Hoffmans, R., Pascual, J., Imamura, S., Kishi, S., Amatruda, J.F., Kanki, J.P., Green, D.R., D'Andrea, A.A., and Look, A.T., Chk1 Suppresses a Caspase-2 Apoptotic Response to DNA Damage that Bypasses p53, Bcl-2, and Caspase-3, 864-877, Copyright (2008) with permission from Elsevier. Full text @ Cell