Cells and treatments
IMR90 and H460 cells were obtained from ATCC. Control and p53−/− HCT116 cells were a gift from B. Vogelstein (Johns Hopkins University). H460, IMR90 and HCT116 derivatives were grown in RPMI-10% serum, DMEM-15% serum and F12/DMEM-10% serum media, respectively. Seckel syndrome fibroblasts (GM18366, Coriell) were propagated in DMEM-15% serum. Primary human bronchial epithelial cells were grown as recommended by a supplier (Clonetics). With the exception of experiments in , cells were treated with FA for 3 h in their complete media. Inhibitors were typically added for 1 h before FA exposure. IMR90 cells were put into a quiescent state by growing them to a full confluency and then maintaining for 2 d in 0.5% serum.
shRNA and siRNA
For stable expression of shRNA, cells were infected with the constructs based on the pSUPER-RETRO vector. The targeting sequences were GACTCCAGTG-GTAATCTAC for p53 and GGTGATATGAAACTCTACT and GACCTGTTATGGAATTTGA for XPA. The shRNA sequence for MSH2 was reported earlier.68
Packaging, infection and selection conditions were used as described previously.69
For ATR knockdown by siRNA, ON-TARGETplus (CGAGACUUCUGCGGAUUGCdTdT; GAACAACACUGCUGGUUUGUU) and control siRNAs were purchased from Dharmacon. H460 cells were seeded to obtain ~25% confluency on the day of transfection. Transfections were performed twice with 50 nM siRNA (final concentration) using Lipofectamine RNAimax (Invitrogen). Cells were treated with FA 48 h after initial transfection.
Western blotting and immunoprecipitation
Cellular extracts were prepared as described earlier.36
Proteins were separated by SDS-PAGE and electrotransferred to ImmunoBlot PVDF membrane (BioRad). Primary antibodies used were: p53 (DO-1, Santa Cruz), ATR (N-19, Santa Cruz), PARP (9542, Cell Signaling), pS15-p53 (9284, Cell Signaling), p21 (SX118, BD Biosciences), CHK1 (2345, Cell Signaling), pS317-CHK1 (2344, Cell Signaling), pS345-CHK1 (2341, Cell Signaling), pT68-CHK2 (2661, Cell Signaling), XPA (556453, BD Biosciences), MDM4 (A300–287A, Bethyl Laboratories) and cyclin A (H-432, Santa Cruz).
In the immunoprecipitation experiments, cells were crosslinked with 1% FA for 3 min followed by quenching with 0.125M glycine in PBS. Cells were then lysed for 5 min on ice in buffer A (10 mM HEPES pH 7.9, 10 mM KCl, 1.5 mM MgCl2, 0.34 M sucrose, 10% glycerol, 1 mM DTT, protease inhibitors) containing 0.1% Triton X-100. After centrifugation (3000 g, 4°C, 5 min), pellets were washed with buffer A and then incubated in buffer B (3 mM EDTA, 0.2 mM EGTA, 1 mM DTT, protease inhibitors) on ice for 5 min. After another centrifugation, insoluble chromatin was washed with buffer B and resuspended in a digestion buffer (10 mM TRIS-HCl pH 7.5, 1.5 mM MgCl2, 10 mM NaCl, 1 mM EDTA, 0.34 M sucrose, 10% glycerol, 0.1% Triton X-100, 10 mM caffeine, 1 μM wortmannin, 100 U/ml benzonase (Sigma) and protease inhibitors) followed by 37°C incubation for 25 min. After digestion, equal volume of binding buffer (100 mM Na2HPO4, 2 mM K2HPO4, 137 mM NaCl, 2.7 mM KCl, 1% Triton X-100, and protease inhibitors) was added, and the lysates were then centrifuged (15,000 g, 4°C, 15 min). Supernatants were incubated with primary antibodies for 2 h at 4°C. Subsequently, 20 μl protein G/A beads (Santa Cruz) were added and incubated for another 1 h before washing three times with 1 ml binding buffer. Samples were boiled in SDS loading buffer for 10 min to reverse crosslinks and antibody binding
Cells were grown on coverslips, treated with FA for 3 h and fixed with 4% paraformaldehyde for 15 min followed by PBS-0.2% Triton X-100 permeabilization for 10 min. S-phase cells were labeled with 10 μM 5-ethynyl-2'-deoxyuridine (EdU) for 15 min. For immunostaining of ATR, RPA32, pS4/8-RPA32 and RAD1, PBS-0.5% Triton X-100 pre-permeabilization was applied before fixation for 10 min on ice. Cells were then blocked with 3% BSA for 1 h followed by EdU staining (Alexa Fluor 647 azide or Alexa Fluor 488 azide) using Click-iT Alexa Fluor EdU Imaging kits (Invitrogen). After EdU staining, cells were incubated with primary antibodies overnight at 4°C. The following primary antibodies and dilutions were used: RAD1 (Q-18 from Santa Cruz, 1:100), RPA32 (RPA34–20 from Calbiochem, 1:300), cyclin B1 (H-433 from Santa Cruz, 1:300), ATR (N-19 from Santa Cruz, 1:200), CDT1 (ab83174 from Abcam, 1:300), pS4/8-RPA32 (Bethyl Laboratories, 1:300) and pS15-p53 (16G8 from Cell Signaling, 1:100). Cells were then washed twice and incubated with secondary antibody for 1 h at room temperature. Images were obtained with Zeiss LSM 710 confocal microscope.
Cells were trypsinized, spun down and then fixed in 1.5% formaldehyde for 10 min followed by ice-cold methanol permeabilization for 10 min. For BrdU immunostaining, cells were incubated with 2N HCl for 20 min. Subsequently, cells were washed twice with 1% BSA in PBS. Samples were incubated with FITC-conjugated anti-BrdU (BD Biosciences) and mouse anti-pS15-p53 (16G8, Cell Signaling) antibodies for 30 min at room temperature. Secondary antibodies for anti-pS15-p53 were Alexa Fluor 488 goat anti-mouse IgG (1:300, Invitrogen). After immunostaining, cells were washed twice and resuspended in 1 ml TE buffer containing
Forty µg/ml propidium iodide and 40 µg/ml RNase for 30 min at 37°C before FACS analysis (FACSCalibur, BD Biosciences). For Annexin V/7-AAD staining, FITC-Annexin V apoptosis staining kit was utilized (BD Biosciences). Cells were harvested and spun down (250 g, 4°C, 4 min) followed by washing twice with PBS and a binding buffer. Cells were then stained with FITC-Annexin V and 7-AAD for 15 min at room temperature followed by immediate FACS analysis.
RNA samples were extracted with Trizol (Invitrogen), purified with RNeasy mini kit (Qiagen) and further treated with DNase I. cDNA was then synthesized by RT First Strand kit (SABioscience) per manufacturer’s protocol. Primers were obtained from SABioscience and amplification reactions were performed using ABI 7900HT Real-Time PCR system (Applied Biosystems). Expression of five housekeeping genes (B2M, HPRT1, RPL13A, GAPDH, ACTB) were used for normalization purposes. Fold differences of gene expression were calculated by the 2-DDCt method.
Senescent cells were identified by staining for senescence-associated β–galactosidase activity (SA-β-Gal assay).70
IMR90 cells were seeded in 6-well plates, treated with FA and, 3 d later, were replated to avoid over-confluent cultures. On day 6 post-FA, cells were fixed with 0.5% glutaraldehyde in PBS (pH 6.0) for 5 min, washed twice with PBS and then incubated with a β-galactosidase staining solution adjusted to pH 6.0 (Roche) at 37°C for 16 h. To facilitate counting of cells, 100 ng/ml DAPI was added to stain nuclei. Five fields were randomly selected to count at least 100 cells for each well.