The phosphorylation of Atr substrates are diminished in Bid-deficient cells following replicative stress. (a) Phosphorylated mouse Chk1 presents as a shifted band. Bid+/+ and Bid−/− MPCs were treated with 10 mM HU for 2 h. Whole-cell extracts were incubated with 10 U Calf Intestinal Alkaline Phosphatase (Invitrogen)/100 μg lysate, resolved by SDS-PAGE and immunoblotted with anti-Chk1, anti-pChk1 (S317), or anti-pChk1 (S345) as indicated. Solid arrows denote the mobility of shifted phosphorylated Chk1, and dashed arrows denote the mobility of unphosphorylated Chk1. (b) Bid+/+ and Bid−/− MPCs were treated 10 mM HU for the indicated times. Total cell lysate was resolved by SDS-PAGE followed by immunoblotting with the indicated antibodies. (c) U2OS cells were treated with Bid-specific siRNA no.7, Bid-specific siRNA no.8, or control siRNA for 72 h. Bid KD and control KD cells were treated with 10 mM HU or 25 μM ETOP for 2 h, and total cell lysate was resolved by SDS-PAGE followed by immunoblotting with the indicated antibodies. (d) U2OS cells transfected with control siRNA or Bid siRNA (no. 8) for 72 h were treated with 10 mM HU for 2 h. Whole-cell lysates were immunoprecipitated with anti-Chk1 antibody, and the immunoprecipitated product was incubated with 1 μg GST-Cdc25C protein, 10 μM cold ATP and 5 μCi γ-³²P-ATP in kinase buffer. Chk1 kinase reactions were resolved on SDS-PAGE, stained with SimplyBlue SafeStain (Invitrogen) to visualize GST-cdc25c levels, and analyzed by autoradiography. (e) Bid+/+ and Bid−/− MPCs were treated 10 mM HU over time. Total cell lysate was resolved by SDS-PAGE followed by immunoblot with the indicated antibodies. Relative band intensity has been measured by densitometry analysis. (f) U2OS cells were transfected with control siRNA or Bid siRNA for 72 h, and then treated with 10 mM HU over time. Total cell lysate was resolved by SDS-PAGE followed by immunoblot with the indicated antibodies. Solid arrow denotes the mobility of shifted phosphorylated RPA32, and dashed arrow denotes the mobility of unphosphorylated RPA32. Relative band intensity was measured by densitometry analysis

The helix 4 domain of Bid interacts with the coiled-coil domain of Atrip. (a) Schematic illustration of mouse Bid structure. (b) Cells (293T) were co-transfected with HA-Atrip/pLPCX, and either wild-type Bid, or Bid mutated in the BH3 domain or Bid mutated in the Atm/Atr consensus phosphorylation sites. Bid was immunoprecipitated from whole-cell extracts using anti-Bid antibody. Samples were resolved on SDS-PAGE followed by immunoblotting with the indicated antibodies. (c) Sequence alignment of the helix 4 and helix 5 of Bid among different species. Helix 4 and helix 5 are labeled as purple arrows. The GenBank accession numbers of the sequences used here are: Homo sapiens (NM_197966), Mus musculus (NM_007544), Rattus norvegicus (NM_022684), Gallus gallus (NM_204552), Danio rerio (NM_001079826), Sus scrofa (NM_001030535), Xenopus laevis (NM_001095594), Xenopus tropicalis (NM_001097226), and Bos taurus (NM_001075446). The alignment was performed by Clustal X. The Leu105 and Leu109 in helix 4 are labeled as green stars. The Gln112 and Asn115 in helix are labeled as cyan stars. Loop A amino acids are dark blue, Loop B amino acids are pink. The same amino acids are labeled in the nuclear magnetic resonance structure of Bid.³⁰ Helix 4 is denoted in red, and is on an exposed surface of Bid. The BH3 domain is facing out of the page. (d) Cells (293T) were co-transfected with HA-Atrip/pLPCX, and wild-type Bid or Bid harboring mutations in helix 4: mutation of green stars to polar cysteine residues (H4A), or of cyan stars to alanine residues (H4B), or of dark blue stars at the end of helix 4 to alanine residues (loop A), or of pink stars in the loop region between helices 4 and 5 to glycine (loop B) Bid was immunoprecipitated from whole-cell extracts and samples were analyzed as above. (e) Wild-type or helix 4-mutated Bid and His-MBP-Atrip protein were purified from E.coli. Bid (10 μg) and 100 μg His-MBP-Atrip protein were incubated in binding buffer at room temperature for 30 min. Bid was immunoprecipitated using anti-Bid antibody, and the immunoprecipitated proteins were resolved on SDS-PAGE and immunoblotted with the indicated antibodies. (f) Schematic illustration of Atrip structure. CRD, checkpoint recruitment domain. CC, coiled-coil domain. TopBP1, TopBP1-interacting domain. ATR, Atr-binding domain. (g–i) Wild-type Bid and HA-tagged full-length or various truncated Atrip constructs were overexpressed in human 293T cells. Then the cells were treated with 10 mM HU for 2 h and Bid was immunoprecipitated by anti-Bid antibody. The immunoprecipitated products were detected by anti-Bid and anti-HA antibodies

An intact Bid helix 4 is required for Bid's function following HU treatment. (a) U2OS cells transfected with control siRNA or Bid siRNA, and Bid KD cells with rescue by wt-Bid, H4A, H4B or phospho-mutated (S/A) Bid were treated with 10 mM HU for 5 h, fixed, and stained with anti-Atrip antibody. Representative images of Atrip staining were shown. (b) Quantitative analysis of Atrip accumulation at nuclear foci following replicative stress. The percentage of cells with >5 clearly visible Atrip nuclear foci was calculated for each cell type. More than 600 cells were counted in three independent experiments. (c) U2OS cells were treated with control siRNA or Bid siRNA for 72 h. Wild-type mouse Bid, Bid H4A, Bid H4B, or vector alone was introduced into the cells simultaneously with siRNA. Cells were treated with 10 mM HU for 2 h. Total cell lysate was resolved on SDS-PAGE and immunoblotted as above. Relative band intensity of pChk1 signal has been measured by densitometry analysis. (d) U2OS cells overexpressing HA-tagged wild-type or helix 4-mutated hBid was transfected with Bid siRNA for 72 h. Silent mutations were introduced in the Bid siRNA-target region so that only endogenous Bid was knocked down by Bid siRNA. Then, cells were treated with HU overnight. The untreated and treated cells were collected in ice-cold PBS and detected in alkaline comet assay. At least 60 randomly chosen comets/sample were analyzed by CometScore Program Version 1.5. ^*P<0.05. (e) U2OS cells overexpressing HA-tagged wild-type or helix 4-mutated hBid was transfected with Bid siRNA for 72 h. Silent mutations were introduced in the Bid siRNA-target region so that only endogenous Bid was knocked down by Bid siRNA. Then, cells were treated with HU for overnight and released into fresh media containing 1 μg/ml nocodazole for the indicated times. Cells were fixed and stained with propidium iodide. Live cells were gated on FSC/SSC and analyzed by flow cytometry. The quantitative analysis of the arrested G1/early S phase cells following 32 h HU withdrawal was shown in (f). ^*P<0.05. (f) The quantitative analysis of the arrested G1/early S-phase cells following HU withdrawal in Figure 6e. Data were collected from three independent experiments. ^*P<0.05

The cellular recovery from replicative stress and the HU-induced accumulation of Atrip at nuclear foci are impaired in the absence of Bid. (a) Bid is highly expressed in hematopoietic tissues. Tissues were harvested from wild-type C57Bl6 mice. Total cell lysates from the indicated tissues were resolved by SDS-PAGE and immunoblotted with the indicated antibodies. The molecular weight markers used in immunoblots are labeled on the right of the blots. (b) Bid−/− bone marrow cells are more sensitive to replicative stress. Bid+/+ and Bid−/− mice were injected with 100 mg/kg hydroxyurea (HU) for 3 consecutive days. Mice were killed and bone marrow was harvested from mouse femurs and tibia at 24 h after the third injection. Bid+/+ and Bid−/− mice were irradiated with 2 Gy using a ¹³⁷Cs source. Mice were killed and bone marrow was harvested from mouse femurs and tibia 24 h after irradiation. Following lysis of red blood cells, viable bone marrow cells were identified by trypan blue exclusion and counted. N=15 mice for HU treatment and n=10 mice for irradiation treatment. Error bar=90% confidence interval. P-value is calculated by student's t-test. (c) U2OS cells were transfected with control siRNA or Bid siRNA. After 2 days, cells were exposed to 10 mM HU for 24 h, and released into fresh media containing 1 μg/ml nocodazole for the indicated times. Cells were fixed and stained with 7-AAD and analyzed by flow cytometry. (d) U2OS cells were transfected with control siRNA or Bid siRNA for 72 h. Cells were lysed and Bid was detected in immunoblots. (e) Bid+/+ and Bid−/− MPCs were treated with 10 mM HU for 2 h. The chromatin fraction was isolated and extracts were resolved on SDS-PAGE and immunoblotted with the indicated antibodies. (f) U2OS cells were transfected with control siRNA or Bid siRNA and wild-type mouse Bid was introduced into the cells simultaneously with siRNA. Cells were treated with 10 mM HU for 5 h, fixed, and stained with anti-Atrip antibody. Representative images of Atrip staining were shown. (g) Quantitative analysis of Atrip accumulation at nuclear foci following replicative stress. The percentage of cells with >5 clearly visible Atrip nuclear foci was calculated for each cell type. More than 600 cells were counted in three independent experiments

Bid associates and co-localizes with Atr/Atrip/RPA complex following replicative stress. (a) Bid+/+ and Bid−/− MPCs were treated with 10 mM HU for 2 h. Bid was immunoprecipitated from nuclear extracts using biotin-conjugated anti-Bid antibody and streptavidin–agarose beads. Samples were analyzed using SDS-PAGE followed by immunoblotting with the indicated antibodies. The asterisk (^*) indicates a crossreacting band. Transcription factor RUNX1 was used as a negative control. (b) U2OS cells were treated with 10 mM HU for 2 h. Cells were harvested, and Atrip was immunoprecipitated from nuclear extracts using anti-Atrip (401) antibody. Immunoprecipitates were analyzed using SDS-PAGE followed by immunoblotting with anti-Bid and anti-Atrip antibodies. (c) U2OS cells were treated with 10 mM HU for 2 h. Cells were harvested, and RPA was immunoprecipitated from nuclear extracts using anti-RPA70 antibody. Immunoprecipitates were resolved by SDS-PAGE followed by immunoblotting with anti-Bid and anti-RPA70 antibodies. (d) The interaction between Bid and Atr complex is independent of DNA. Bid+/+ MPCs were treated with 10 mM HU for 2 h. Then, the nuclear fraction was purified and incubated with 250U Benzonase Nuclease (Novagen). Then, Bid was immunoprecipitated from nuclear extracts using biotin-conjugated anti-Bid antibody and streptavidin–agarose beads. Samples were analyzed using SDS-PAGE followed by immunoblotting with the indicated antibodies. (e) Bid−/− MEFs harboring HA-tagged Bid were synchronized in low serum medium (0.1% FBS–DMEM) for 24 h. Following synchronization, cells were released into complete medium (10% FBS–DMEM). At 17 h after release, cells were left untreated (18 h serum) or treated for 1 h with 1 mM HU (18 h serum plus HU). Then, cells were fixed and stained for anti-HA and anti-RPA32 antibodies. Representative images in (f) were captured by a Zeiss LSM 510 inverted confocal microscopy. Scale bars represent 10 μm

Mutations in helix 4 domain of Bid do not significantly change the function of Bid in the extrinsic cell death pathway. (a) Helix 4 mutated Bid binds with other Bcl-2 family proteins. Cells (293T) were transfected with wild-type Bid or helix 4-mutated Bid. Bid was immunoprecipitated from whole-cell extracts. Immunoprecipitates were resolved by SDS-PAGE followed by immunoblotting with anti-Bid, anti-Bcl-2 and anti-Mcl-1 antibodies. All the samples were run in the same gel with interrupted lanes deleted. (b) Helix 4-mutated Bid show similar sensitivity as wild-type Bid to caspase 8. Purified wild-type and two helix 4 Bid mutants (H4A and H4B) were cleaved by active caspase 8 (Millipore) in vitro for 0.5 and 1 h. The full-length and truncated Bid in reaction products were analyzed by anti-Bid antibody in immunoblots. (c) U2OS cells overexpressing HA-tagged wild-type or helix 4-mutated Bid was transfected with Bid siRNA for 72 h. Silent mutations were introduced in the Bid siRNA-target region so that only endogenous Bid was knocked down by Bid siRNA. Then, cells were treated with 50 ng/ml TRAIL and 5 μg/ml cycloheximide (CHX) for 4 h. Total cell lysate was analyzed by SDS-PAGE followed by immunoblotting with anti-Bid antibody. Solid and dashed arrows denote endogenous Bid and overexpressed Bid, respectively. (d) Cells harboring helix 4-mutated Bid show similar sensitivity to TRAIL/CHX treatment. U2OS cells overexpressing HA-tagged wild-type or various Bid mutations was transfected with Bid siRNA for 72 h. Then, cells were treated with 50 ng/ml TRAIL and 5 μg/ml CHX over time. The apoptotic cells were detected by Annexin V-FITC Apoptosis Detection Kit (BioVision, Mountain View, CA, USA)

A proposed model for Bid in the Atr-mediated DNA damage response to replicative stress. (a) The damage complex is unstable in Bid−/− MPCs following HU treatment. Bid+/+ and Bid−/− MPCs were treated with 10 mM HU for 2 h. Then, the nuclear fraction was purified and RPA or Atr was immunoprecipitated from nuclear extracts. The immunoprecipitated samples were analyzed using SDS-PAGE followed by immunoblotting with the indicated antibodies. (b) Schematic model of a proposed role for Bid in the Atr-mediated DNA damage signaling pathway. Bid serves as a mediator in the Atr-directed response to replicative stress at the level of Atr/Atrip activation. Bid associates with the Atr/Atrip complex via Atrip. Thus, Bid functions at the level of the sensor complex, to facilitate and amplify Atr-directed Chk1 activation and ensure rapid and efficient checkpoint activation