A DNA-PK phosphorylation site on MET regulates its signaling interface with the DNA damage response

Inhibitors

If not indicated otherwise, tepotinib/EMD1214063 (Merck KGaA, Darmstadt, Germany) was dissolved in DMSO and used at a final concentration of 50 nM. KU55933 (ATM inhibitor), VE-821 (ATR inhibitor) and KU57788 (PRKDC (DNA-PK) inhibitor) (all Selleck Chemicals, Houston, Texas, USA) were used at a final concentration (f.c.) of 10 µM if not specified otherwise. DNA-PK inhibitor peposertib/M3814 (Merck KGaA, Darmstadt, Germany) was used at final concentrations of 100 nM and 300 nM. Inhibitors were dissolved in DMSO and working solutions were prepared freshly and remained in the media for the duration of the respective experiment.

In vitro kinase assay

PRKDC (DNA-PKcs) in vitro kinase assay on synthetic peptides was performed using the ADP-GloTM system (#V4107, Promega, Madison WI, USA) along with the DNA-PKcs Kinase Enzyme System (#V4106, Promega) according to manufacturer’s instructions. The following peptides corresponding to the MET Ser1016 region were used as substrates: DQFPNSSQNG (“WT”), DQFPNSSANG (“Q-A”), DQFPNASQNG (“S1-A”) DQFPNSAQNG (“S2-A”), DQFPNAAQNG (“S1S2-AA”). As a control for the reactions, we used peptides corresponding to Ser139 region of H2AX: KKATQASQEY (“WT”), KKATQASAEY (“Q-A”), and KKATQAAQEY (“S-A”). Each reaction condition was independently set up and measured three times.

Cell lines maintenance

Human gastric carcinoma cell line GTL-16 (provided by Dr. Paolo Comoglio (Medical School University of Torino, Italy)) and the non-small cell lung cancer cell line EBC-1 (provided by Dr. Silvia Giordano (University of Torino, Torino, Italy)) were cultured in RPMI medium (GIBCO, Invitrogen) supplemented with 5% and 10% FCS, respectively, and antibiotic-antimycotic (penicillin 100 U/mL, streptomycin sulfate 100 U/mL, amphotericin B 0.25 mg/mL; Gibco). NIH 3T3 cells (provided by Dr. Laura Schmidt (NCI, Bethesda, MD, USA)) were grown in DMEM medium (Gibco) supplemented with 10% FCS (Sigma), antibiotic-antimycotic (penicillin 100 U/mL, streptomycin sulfate 100 U/mL, amphotericin B 0.25 mg/mL; Gibco) and puromycin (Sigma, 1.5 μg/mL). Profiling of the EBC-1 cell line was done by using highly polymorphic short tandem repeat loci in June 2020 (Microsynth), the GTL-16 cells and NIH 3T3 mutants have been authenticated by whole-exome sequencing and transcriptomic analysis. All cell lines have been regularly tested for mycoplasma contamination.

siRNA-mediated knockdown of MET, ATM, ATR, and DNA-PK

siRNAs targeting MET (Cat. # L-003156-00-0005), ATM (Cat # L-003201-00-0005), ATR (Cat. # L-003202-00-0005), DNA-PKcs (Cat #L-005030-00-0005), and the non-targeting siRNA control pool (Cat. #D-001810-10-20) were purchased from Dharmacon.

siRNAs were transfected using TransIT-X2® Dynamic Delivery System (Cat. #MIR6004) according to manufacturer’s instructions.

After seeding and adherence for 24 h, EBC-1 cells were transfected with 25 nM MET, ATM, ATR, and DNA-PK siRNAs or with 25 nM of non-targeting siRNA control. The cells were harvested for western blot analysis 48 h (non-targeting control, MET, ATM, ATR) or 96 h (DNA-PK) post siRNAs transfection.

After seeding and adherence for 24 h, GTL-16 cells were transfected with 25 nM MET siRNA or 50 nM DNA-PK siRNA and with non-targeting siRNA control, 25 nM or 50 nM, respectively. The cells were harvested for western blot analysis either 48 h (control siRNA and MET siRNA) or 72 h (control siRNA and DNA-PK siRNA) post siRNAs transfection.

Plasmids and NIH-3T3 cells transfections

Site-directed mutagenesis was performed with the QuikChange Lightning kit (Stratagene) according to the manufacturer’s instructions. The mutations described in the main text were inserted into the pBabe puro c-met WT plasmid (gift from Joan Brugge, Addgene plasmid #17493; http://n2t.net/addgene:17493; RRID:Addgene_17493) [49]. NIH 3T3 cells were transfected with Lipofectamine™ 2000 (Invitrogen) and clones were selected with puromycin (Sigma). In all the experiments, clonal cell lines derived from a single cell each have been used. Clones expressing identical levels of total MET as well as equal basal MET Y1234/5 autophosphorylation have been employed (Fig. S5).

Western blotting and antibodies

Protein extracts were prepared as previously described [34], separated by SDS-PAGE and blotted onto PVDF membranes. After incubation with primary antibodies (see below), signal was detected using fluorescent secondary antibodies (LI-COR). Antibodies used: pS1016 MET (CST, custom-made rabbit polyclonal, see Fig. S1 for data on antibody validation), β-Actin (Millipore, MAB1501) total MET (CST, 3127), p-Y1234/5 MET (CST, 3126), p-AKT (CST, 9271), p-ERK1/2 (CST, 4370), p-S6 (CST, 4858), pH3 (Millipore, 06-570), γH2AX (Millipore, 05-636), pChk1 (CST, 2341), pChk2 (CST, 2661). Blots were quantified with ImageStudio Lite 5.2.5. All experiments were performed independently at least three times, representative blots are shown.

Proliferation assay

Cells were plated in triplicates in 24-well plates (300 cells/well) and treated as indicated one day after plating. Six days after treatment, cells were fixed and stained with 2% crystal violet (Sigma) in acetic acid-methanol (2:1). Cell density was measured with ImageJ. Experiments were repeated three times.

Viability assay

Cells were plated in triplicates in 24-well plates (300 cells/well) and treated as indicated one day after plating. Six days after treatment, cells were incubated with resazurin blue (Sigma, final concentration: 3 µM) for two hours before measuring the fluorescent signal (excitation: 545 nm, emission: 590 nm). Experiments were performed in three biological replicates.

Live/Dead assay

The assay was performed as recommended by the manufacturer (ThermoFisher). Briefly, cells were stained with Calcein AM (live cells, green) and ethidium homodimer-1 (dead cells, red) 48 h after a 2 Gy irradiation. Cells were imaged with a fluorescence microscope (Leica) and analyzed with ImageJ. Experiments were repeated independently three times.

Mouse xenograft

20’000 cells of the indicated cell lines were injected into the flank of immunocompromised Rag2-/- yc-/- mice (8-12-week-old). Tumor growth was followed daily by caliper measurement of the width, length, and depth (raw tumor sizes on each day for each experimental animal are provided in Table S2). Once tumors had reached the appropriate size, mice were randomly attributed to the control or treatment (local single-dose 6 Gy irradiation using XStrahl 150 (XStrahl Limited)) groups. Each group included 3 males and 3 females. Investigators performing treatments and tumor size measurements were blinded to group allocation. Animal experiments were approved by the local experimental animal committee of the Canton of Bern and performed according to Swiss laws for animal protection (animal license nr. BE13/13).

Comet assay

Comet assay was performed according to the manufacturer’s instructions (Trevigen), tail intensity was measured for 50 cells/condition with the Comet assay IV program (Andor Technology). Experiments were repeated independently three times.

Fluorescence microscopy

Cells were plated on 8-chamber microscopy slides and treated as indicated. Cells were fixed with 4% formaldehyde (Sigma), permeabilized with 0.2% Triton-X100 (Sigma) and blocked with 3% goat serum (Dako). After incubation with the appropriate primary antibody, the signal was detected with fluorescent secondary antibodies (ThermoFisher), the cells were counterstained with DAPI (Sigma, 300 nM). The coverslips were mounted with Vectashield antifade mounting medium (Vector laboratories). Imaging was performed with a fluorescence microscope (Leica). γH2AX foci formation was quantified with CellProfiler. Antibodies used: γH2AX (CST, 9027 S), α-tubulin (Sigma, T6199), γ-tubulin (Sigma, T3559). The quantification and statistical analysis of 100 cells per condition (γH2AX foci) or a triplicate of experiments (cell division) is shown.

Apoptosis assay

Apoptosis induction was measured by flow cytometry with a FITC-Annexin V/propidium iodide kit according to the manufacturer’s instruction (Invitrogen). Acquisition was performed on an LSR II (BD Biosciences) and analysis was performed with FlowJo (FlowJo, LLC). The quantification and statistical analysis of a triplicate of experiments is presented.

Cell cycle analysis

Cell cycle distribution was measured by flow cytometry. At the appropriate timepoint after treatment, cells were collected and fixed in 70% EtOH, washed with PBS and stained with a propidium iodide (20 μg/mL, Sigma)-RNAse A (40 μg/mL, Qiagen)-Triton-X100 (0.2%) solution in PBS. Acquisition was performed on an LSR II (BD Biosciences) and the data were analyzed with FlowJo (FlowJo, LLC). Experiments were performed three times.

CFSE dye dilution assay

Cells were stained according to the manufacturer’s instructions (CellTrace CFSE, Invitrogen) before plating. One day after plating, cells were treated as indicated. Samples were collected daily for 5 days, starting on the day of treatment. Signal intensity was measured by flow cytometry with an LSR II (BD Biosciences) and analysis was performed with FlowJo (FlowJo, LLC). Signal intensity was plotted as a function of time, and the proliferation rate was obtained by calculating the slope of the linear regression of this function. The statistical analysis was performed on a biological triplicate of experiments.

Discovery phosphoproteomics

Cell cultures were washed, scraped in phosphate-buffered saline, and spun down for 5 min at 1000 rpm. Resulting pellets were resuspended in 8 M urea solution containing 0.1 M ammonium bicarbonate and disrupted by sonication. Supernatants were centrifuged at 12000 rpm for 10 min and protein concentration was determined by BCA Protein Assay (Pierce). Disulfide bonds were reduced with tris(2-carboxyethyl)phosphine at a final concentration of 5 mM at 37 °C for 30 min and alkylation of free thiols was performed with 10 mM iodoacetamide at room temperature for 30 min in the dark. The solution was subsequently diluted with 0.1 M ammonium bicarbonate to a final concentration of 1.5 M urea and digestion was done overnight at 37 °C by sequencing-grade modified trypsin (Promega) at a protein-to-enzyme ratio of 50:1. Acidification was performed by adding formic acid to a final pH <3 to stop protein digestion. Peptides were desalted on a C18 Sep-Pak cartridge (Waters) and one-tenth of the resulting eluate was processed individually for total proteome analysis. Phosphopeptides were enriched from 1 mg of initial peptide mass with TiO2 as previously described [50]. For mass spectrometry analysis, samples were resuspended in 20 μl of 2% acetonitrile, 0.1% formic acid, and 1 μl of each sample was used for injections. LC-MS/MS analysis was performed with an Easy nLC 1000 system (Thermo) connected to an Orbitrap Elite mass spectrometer (Thermo) equipped with a NanoFlex electrospray source. Peptides were separated on an Acclaim PepMap RSLC C18 column (150 mm × 75 μm, 2 μm particle size, Thermo) using a gradient of 5–30% buffer B (98% acetonitrile, 2% water, 0.15% formic acid) over 180 min at a flow rate of 300 nl/min. The Orbitrap Elite was operated in data-dependent acquisition mode, each cycle consisting of one MS scan followed by 15 MS/MS scans of the most abundant precursor ions. Collision-induced dissociation was performed with the following settings: isolation width, 2 m/z; normalized collision energy, 35; activation time, 10 ms. Acquired MS data files were subsequently processed for identification and quantification using Maxquant version 1.5.2.8. Settings were kept as default with the following specifications: “First search peptide tolerance” was set to 50 ppm and “Main search peptide tolerance” to 10 ppm. Variable modifications considered were oxidation (Met) and phosphorylation (Ser/Thr/Tyr). “Label free quantification” and “Match between runs” were enabled, with a match time window of two minutes. The search was performed against the mouse UniProt FASTA dataset UP000000589. Differential expression analysis of data from the phosphoproteomics measurements was done using the Python package ProtRank [35]. The input data comprised raw counts of 7572 phosphopeptides that have been measured in 4 samples (4 cell lines, 3 conditions: control, 1 h and 7 h after 10 Gy; two replicates for each sample). Of all raw counts, 44% were missing values (zeros). The thresholding for significantly differentially expressed phosphopeptides was done using the false detection rate (FDR) of 0.25 (unless specified otherwise). The subsequent enrichment analysis of the obtained phosphopeptides was performed using STRING (https://string-db.org/) [51]. The results are visualized as network maps: every protein is a “node”, and the predicted associations between proteins are represented by “edges” (lines of varying thickness according to the confidence of the predicted interaction). Relevant enrichments are highlighted in colors as indicated in the figure legend.

Senescence β-galactosidase assay

Cells were plated in 6-well plates and treated as indicated. Seven days after treatment, cells were stained with X-Gal as previously described [52] and imaged with an inverted microscope. Representative pictures from three independent experiments are presented.

Irradiation

Cells were irradiated using a Cesium137 source at a dose rate of 0.86 Gy/min (Gammacell 40, MDS Nordion). For the experiments assessing γH2AX foci formation, cells were irradiated by X-Rad225XL (Precision X-Ray) at a dose rate of 116.2cG/min and employing the 0,3 mm Cupper filter.

Multiple sequence alignment

The alignment was performed on uniprot.org with the default clustalo settings: The default transition matrix is Gonnet, gap opening penalty is 6 bits, gap extension is 1 bit. Clustal-Omega uses the HHalign algorithm and its default settings as its core alignment engine. The algorithm is described in ref. [53].

Statistical analysis

Statistical analysis was performed using Prism 7.03 (GraphPad), p-values < 0.05 were considered significant (*p < 0.05; **p < 0.01; ***p < 0.001). If not stated otherwise, all the experiments were performed at least three times.

留言 (0)

沒有登入
gif