Lead contact and materials availability
Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact Matthias Dobbelstein (email@example.com).
This study did not generate unique reagents.
Experimental model and subject details
The human osteosarcoma cell line U2OS (p53 proficient, female) was purchased from ATCC (RRID:CVCL_0042). Cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (Merck), 2 mM l-glutamine (Life Technologies), 50 units/ml penicillin, 50 μg/ml streptomycin (Gibco), and 10 µg/ml Ciprofloxacin (Bayer) at 37 °C in a humidified atmosphere with 5% CO2. Cells used were routinely tested and ensured to be negative for mycoplasma contamination.
Treatments and transfections
Cells were treated with thapsigargin (Thap, Sigma), 1H-Benzimidazole-1-ethanol, 2,3-dihydro-2-imino-alpha-(phenoxymethyl)-3-(phenylmethyl)- monohydrochloride (BEPP, Sigma), l-Histidinol (l-Hist, Sigma), (E)-2-(2-Chlorobenzylidene) hydrazinecarboximidamide (Sephin, Sigma), trans–N,N′-(Cyclohexane-1,4-diyl)bis(2-(4-chlorophenoxy)) acetamide (Integrated stress response inhibitor or ISRIB, Sigma), GSK2606414 (PERK inhibitor or PERK i, Calbiochem), gemcitabine (Gem, Actavis), Cycloheximide (CHX, Sigma), 5,6-Dichloro-1-β-d-ribofuranosylbenzimidazole (DRB, Sigma), or LDC067 (Selleckchem) as indicated in the figure legends. Thap, BEPP, Sephin, ISRIB, PERK i, DRB, and LDC067 were dissolved in DMSO, l-Hist, and gemcitabine dissolved in water, and CHX was dissolved in 100% ethanol.
siRNA transfections were performed using Lipofectamine 3000 (Life Technologies). Cells were reverse transfected with 100 nM siRNA against SLBP (Ambion, custom made, pool of 3 siRNAs) or negative control scrambled siRNA (Ambion, pool of 2 siRNAs), medium replenished after 24 h and cells harvested 40 h post-transfection. For plasmid overexpression, 2 µg of the respective plasmids were forward transfected using Lipofectamine 2000. Medium was replenished after 6 h, and cells were harvested for experiments 24 h post-transfection. The following plasmids were used.
To obtain a majority population of cells in S phase, cells were synchronized using double thymidine block. Briefly, cells were seeded accordingly and allowed to settle and attach onto plates or coverslips for at least 6 h, then treated with 2 mM thymidine (Sigma). After 16 h, cells were washed once in PBS and then replenished with fresh DMEM for 8 h prior to the second thymidine block (2 mM) for another 16 h. Depending on the assay, cells were released into fresh DMEM for 1 h (celigo proliferation assay) or 4 h (R-loop detection on cells treated with CHX) prior to treatment, harvest and analysis.
Cells were washed once in PBS and harvested in radioimmunoprecipitation assay (RIPA) lysis buffer (20 mM TRIS-HCl pH 7.5, 150 mM NaCl, 10 mM EDTA, 1% Triton-X 100, 1% deoxycholate salt, 0.1% SDS, 2 M urea) in the presence of protease inhibitors. Samples were briefly sonicated to disrupt DNA-protein complexes. The protein extracts were quantified using the Pierce BCA Protein assay kit (ThermoScientific Fisher). Protein samples were boiled at 95 °C in Laemmli buffer for 5 min, and equal amounts were analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Subsequently, proteins were transferred onto a nitrocellulose membrane, blocked in 5% (w/v) non-fat milk in PBS containing 0.1% Tween-20 for 1 h and incubated with primary antibodies at 4 °C overnight followed by incubation with peroxidase-conjugated secondary antibodies (donkey anti-rabbit or donkey anti-mouse IgG, Jackson Immunoresearch). The proteins were detected using either Super Signal West Femto Maximum Sensitivity Substrate (ThermoFisher) or Immobilion Western Substrate (Millipore).
Soluble histones were extracted as described12. Briefly, cells were washed once in PBS and harvested in a low detergent, hypotonic buffer (10 mM Tris, pH 7.4, 2.5 mM MgCl2, and 0.5% NP-40) for 10 min on ice. Following centrifugation at 1000×g, the concentration of the solubilized proteins was determined as described above and equal amounts were analyzed by SDS-PAGE.
|Antibodies||Source (catalog number)||Research resource identifiers (RRID)|
|ATF4 (D4B8)||Cell Signaling (#11815)||RRID:AB_2616025|
|Chk1||Cell Signaling (#2360)||RRID:AB_2080320|
|eIF2alpha||Cell Signaling (#9722)||RRID:AB_2230924|
|gamma H2AX, γH2AX (S139)||Cell Signaling (#2577)||RRID:AB_2118010|
|H3K56ac||Cell Signaling (#4243)||RRID:AB_10548193|
|H4K5ac (EP1000Y)||Abcam (ab51997)||RRID:AB_2264109|
|H4K12ac (EPR17906)||Abcam (ab177793)||RRID:AB_2651187|
|HSC70||Santa Cruz (sc-7298)||RRID:AB_627761|
|phospho-Chk1 (S317)||Cell Signaling (#2344)||RRID:AB_331488|
|phospho-eIF2alpha (S51)||Cell Signaling (#9721)||RRID:AB_330951|
|SLBP (EPR12673)||Abcam (ab181972)||N/A|
DNA fiber assay
DNA fiber assays were performed as described previously9. Briefly, cells were incubated with 5-chloro-2′-deoxyuridine (CldU, Sigma-Aldrich) for 30 min, followed by 60 min incubation with 5-iodo-2′-deoxyuridine (IdU, Sigma-Aldrich) in the presence of inhibitors or treatments as indicated. For the 7-label assay, cells were incubated with CldU for 1 h and then pulsed labeled with IdU and CldU for 15 min each for a total duration of 1.5 h.
Cells were lysed using spreading buffer (200 mM Tris pH 7.4, 50 mM EDTA, 0.5% SDS) and DNA fiber spread on glass slides prior to fixation in a methanol:acetic acid solution (3:1). Upon treatment with 2.5 M HCl, fibers were incubated with rat anti-BrdU antibody (Abcam, RRID:AB_305426, 1:1000, to detect CldU) and mouse anti-BrdU (Becton Dickinson, RRID:AB_10015219, 1:400, to detect IdU) for 1 h at room temperature, then fixed with 4% paraformaldehyde in PBS for 10 min. Slides were incubated with Alexa Fluor 555-conjugated goat anti-rat IgG antibody (RRID:AB_141733) and Alexa Fluor 488-conjugated goat anti-mouse IgG antibody (RRID:AB_138404) (both from ThermoFisher, 1:200) for 2 h at room temperature.
Cells were seeded on glass coverslips, transfected or treated with reagents accordingly and fixed with 4% paraformaldehyde in PBS for 10 min. Then, cells were permeabilized with 0.5% Triton-X 100 in PBS for 15 min, blocked with 3% bovine serum albumin (BSA) in PBS containing 0.1% Tween-20 for 1 h and incubated overnight at 4 °C with S9.6 antibody (Kerafast, RRID:AB_2687463, 1:100, to detect DNA:RNA hybrids). Coverslips were washed in PBS prior to incubation with Alexa Fluor 488-conjugated donkey anti-mouse IgG antibody (ThermoFisher, RRID:AB_141607, 1:250) for 2 h and subsequently counterstained with 0.5 µg/ml DAPI (Sigma) for 5 min prior to mounting using the Fluorescent Mounting Medium from DakoCytomation (#S302380-2) and imaged.
Dot blot analysis
Dot blots were conducted as described previously24. Cells were seeded, treated with Thap, BEPP or CHX as indicated and harvested. Prior to CHX treatment, cells were synchronized using double thymidine block as described (chapter “Cell synchronization”) and released into fresh DMEM for 4 h prior to addition of CHX. Cells were washed once in PBS and fixed with 1.1% paraformaldehyde in a solution of 0.1 M NaCl, 1 mM EDTA, 0.5 mM EGTA, and 50 mM HEPES pH 7 for 30 min at room temperature. To quench the cross-linking reaction, glycin was added to a final concentration of 0.125 M for 5 min. Subsequently, the cells were lysed in 1% Triton-X 100, 0.15 M NaCl, 1 mM EDTA, 0.3% SDS with protease inhibitors. The cell lysates were sonicated for 10 cycles (30 s on/off) (Bioruptor, Diagenode) and then subjected to 2 mg/ml proteinase K (ThermoFisher) treatment for 1 h at 50 °C. DNA was isolated using phenol-chloroform extraction and DNA concentration normalized between samples.
The DNA (1.3 µl) was spotted onto pre-wet nitrocellulose membrane, allowed to air dry and then cross-linked with UVC for 5 min. The membrane was blocked in 5% BSA in PBS containing 0.25% Tween-20 for 30 min at room temperature and subsequently incubated with S9.6 antibody (Kerafast, 1:300) in blocking solution overnight at 4 °C. Following incubation with peroxidase-conjugated donkey anti-mouse IgG (Jackson Immunoresearch, RRID:AB_2340773, 1:10,000), DNA:RNA hybrids (as measured using S9.6 intensity) were detected using Super Signal West Femto Maximum Sensitivity Substrate (ThermoFisher). To confirm the specificity of the antibody, one half of the DNA samples were also pre-treated with RNaseH (0.03 U/ng DNA, Ambion ThermoFisher) for 3 h at 37 °C prior to spotting. As a loading control, the membrane was subsequently incubated with antibodies to single-stranded DNA (ssDNA). Briefly, the membrane was incubated with 2.5 M HCl for 15 min (to denature the DNA), washed with PBS, and incubated with antibody to ssDNA (Millipore, RRID:AB_570342, 1:1000) for 2 h at room temperature. The detection of ssDNA was performed following exposure to secondary antibody using Super Signal West Femto Maximum Sensitivity Substrate (ThermoFisher).
EdU incorporation assay
5-ethynyl-2′-deoxyuridine (EdU, ThermoFisher Scientific, #A10044) was added to exponentially growing cells to a final concentration of 20 µM for 1 h until harvest. Prior to imaging, the cells were fixed and permeabilized as done for immunofluorescence staining. The following reagents were added to 100 mM Na-Phosphate buffer (pH 7) in the following order: 5 µM Alexa Fluor 488 picolyl-azide or 5 µM Alexa Fluor 594 picolyl-azide (Jena Biosciences, #CLK-1276-1 or #CLK-1296-1), 100 µM CuSO4 (Jena Biosciences, #CLK-MI004) in 500 µM tris-hydroxypropyltriazolylmethylamine (THPTA; Sigma-Aldrich, #762342) and 5 mM Na-Ascorbate (Jena Biosciences, #CLK-MI005). The click reaction was performed for 1 h on a shaker, at room temperature and protected from light. Samples were subsequently washed thrice for 10 min with PBS, followed by incubation with 0.3 µg/ml DAPI (Sigma-Aldrich, #D9542) for 10 min. For experiments in Fig. 1, cells were kept in PBS prior to image acquisition with the Celigo Imaging Cytometer (Nexcelom Bioscience). DAPI was used to create a nuclear mask and quantify the DNA content, whereas the nuclear EdU signal was quantified using the Celigo image analysis software. EdU and DAPI signals were presented in a horseshoe plot. For experiments in Fig. 2, coverslips were mounted using the fluorescent mounting medium (DakoCytomation, #S302380-2) and imaged.
Proliferation assay (Celigo)
To study the long-term effect of ISR on cells in S phase, proliferation assay was conducted on synchronized cells. Cells were seeded in technical duplicates in 24-well plates, synchronized using double thymidine block (as described), and released into fresh medium for 1 h then treated with BEPP (30 µM) for 6 h to ensure ISR activation during S phase of the cells. During synchronization, cells were also transfected with plasmids to RNaseH1 or an empty vector control as described previously. After 6 h of treatment, medium was replenished and confluency of cells at day 0 was measured using Celigo Imaging Cytometer (Nexcelom Bioscience). Measurements were made subsequently every 24 or 48 h and medium was changed prior to every measurement.
Quantification and statistical analysis
DNA fiber analysis
To avoid bias, data acquisition and analysis were conducted in a double-blinded manner where identities of the samples were blinded prior to imaging and analysis. Whenever possible, a minimum of 100 DNA fiber structures25 were visualized with fluorescence microscopy (Axio Scope A1 microscope (Zeiss) equipped with an Axio Cam MRc/503 camera) and analyzed.
For the 7-label fiber assay, the number of labels incorporated was counted using the cell counter plugin on Fiji. Fork stalling was then calculated by dividing the number of tracks with less than all seven labels by the total number of tracks and converted into percentage. The length of the second to third label was measured to determine the replication progression for the 7-label fiber assay. The Fiji software (RRID:SCR_002285)26 was used to measure the labeled tracks in pixels and converted to micrometers using the conversion factor of 1 µm = 5.7 pixels (as determined by measuring scale bar under the same microscope settings) and then to kilo base (kb) using the conversion factor 1 µm = 2.59 kb. Rate of fork progression was calculated by dividing the number of bases by the labeling time of the track.
For the 2-label fiber assays, fibers were analyzed for their IdU track length and IdU fork progression rate calculated as described.
A summary of the fiber assay data containing information on the number of fibers sampled, mean, median, and standard deviation of each condition can be found in Supplementary Table 1.
The raw data of each fiber assay showing the analysis conducted (as described above) can be found in Supplementary Table 2.
Nuclear quantification of immunofluorescence
Images were acquired (same exposure time for all images for each fluorescent channel per experiment) with Axio Scope A1 microscope (Zeiss) equipped with an Axio Cam MRc/503 camera.
The Fiji software was used for automated analysis and quantification of nuclear S9.6 or EdU staining. DAPI staining was used to identify regions of interest (nuclei) prior to measuring mean intensity of the Alexa Fluor 488 staining (S9.6), Alexa Flour 488 picolyl-azide or Alexa Fluor 594 picolyl-azide (EdU). At least 200 cells were subjected to analysis and quantification.
Statistical testing was performed using Graph Pad Prism 6 (RRID:SCR_002798). For fiber assay and immunofluorescence experiments where normally distributed data cannot be assumed, Mann–Whitney U test was used to calculate significance. For the other experiments, a two-sided unpaired Student’s t-test was calculated. Significance was assumed where p-values ≤ 0.05. Asterisks represent significance in the following way: ****p ≤ 0.0001, ***p ≤ 0.005; **p ≤ 0.01; *p ≤ 0.05.