Length‐independent telomere damage drives post‐mitotic cardiomyocyte senescence

Ageing studies.

Mixed‐sex C57BL/6 mice were analysed at either 3, 15, 20, 24 or 30 months of age. For whole‐body X‐ray irradiation, mixed‐sex 1‐month‐old C57BL/6 mice were irradiated with 2 Gy followed by 11‐month recovery period before culling. TERC^−/− C57BL/6 mice. Male mice were bred to produce successive generations of mice with decreasing telomere length. Hearts from fourth‐generation (G4) mixed‐sex mice were studied. Catalase^−/− and MnSOD^+/− mice. Mouse models for elevated reactive oxygen species and mitochondrial dysfunction: Catalase^−/− and MnSOD^+/− were aged until 22–24 months. PolgA^mut/mut mice. Mice with a knock‐in missense mutation (D257A) at the second endonuclease‐proofreading domain of the catalytic subunit of the mitochondrial DNA (mtDNA) polymerase Polγ (PolgA^mut/mut mice) and PolgA^+/+ mice were used at 12 months of age. Mice were group‐housed in individually ventilated cages with a constant temperature of 25°C, a 12‐h light/dark cycle and with RM3 expanded chow (Special Diet Services).

For the above mice projects were approved Newcastle University Animal Welfare Ethical Review Board and experiments were conducted in compliance with the UK Home Office (PPL P3FC7C606 or 60/3864).

MAO‐A transgenic mice.

MAO‐A mice, on the C57BL6/J background, with cardiac‐specific overexpression of MAO‐A driven by the α‐MHC promoter are previously described (Villeneuve et al, 2013). Mixed‐sex MAO‐A offspring and non‐transgenic littermates were used for the experiments at 6 months of age. N‐acetyl‐cysteine was provided at 1.5 g/kg/day between 1 and 6 months of age. Mice were housed in a pathogen‐free facility (B 31 555 010). Experiments were approved by University of Toulouse local ethic committee (CEEA‐122 2015‐01) and conformed to the Guide for the Care and Use of Laboratory Animals published by the Directive 2010/63/EU of the European Parliament.

INK‐ATTAC mouse model for the clearance of senescent cells.

Experimental strategy for making transgenic mice with a senescence‐activated promoter coupled to the drug‐activatable ATTAC “suicide” transgene and GFP was devised by JLK and TT. INK‐ATTAC mice were created and characterised at Mayo through a collaboration among the JLK‐TT, NKL and van Deursen laboratories. Animals were crossed onto a C57BL/6 background (van Deursen laboratory) and bred, genotyped and aged (JLK‐TT laboratory). Mixed‐sex mice were housed in a pathogen‐free facility, at 2–5 mice/cage with 12‐h light/12‐h dark cycle at 24°C and ad libitum access to standard mouse diet (Lab Diet 5053, St Louis, MO, USA) and water. AP20187 (10 mg/kg) or vehicle was administered to 27‐month‐old mice by intraperitoneal injection every 3 days for 2 months.

Senolytic treatment of male C57BL/6 mice.

Mice at 22 months of age were purchased from Charles River (Charles River Laboratories International, UK). Mice were randomly assigned to a treatment group. ABT263 (navitoclax) or vehicle alone was administered to mice by oral gavage at 50 mg/kg body weight per day (mg/kg/day) for 7 days per cycle for two cycles with a 1‐week interval between the cycles. Hearts were collected directly into 50 mM KCl to arrest in diastole.

For all animal studies, details of sample sizes are included in the figure legends.

Human tissue collection and ethics.

Human heart tissue was obtained from male and female patients undergoing open‐heart surgery for aortic stenosis, with a section of the right atrial appendage being placed in 10% neutral buffered formalin (VWR, 9713.9010) immediately after dissection. Subsequent processing steps for paraffin embedding were the same as for mouse tissue (as described below). All tissue samples were obtained under the clause in the Human Tissue Act that enables anonymised samples to be taken without consent in the context of an ethically approved study. This study was approved by the Research Ethics Committee, UK, REC reference number: 10/H0908/56.

Adult mouse cardiomyocyte isolation and purification.

Hearts from mixed‐sex CJ57/BL6 mice were placed on a Langendorff setup. Cell suspensions were obtained by enzymatic digestion and sedimentation. To enrich the CM population, cells were stained at 4°C for 30 min with a cocktail of biotin‐conjugated antibodies (CD31 (390), CD45 (30‐F11), and Sca‐1 (D7); BioLegend) and the EasySep™ Mouse Biotin Positive Selection Kit, used to remove the labelled non‐CM cells. Cultured CM purity was assessed by immunofluorescent staining against CD31, Sca‐1, CD45, and α‐actinin. Qiagen RNA extraction kit was used of RNA isolation. CMs were cultured on laminin‐coated wells in MEM with Hank's salts and l‐glutamine (SIGMA), 0.1 mg/ml BSA, 10 mM butanedione monoxime, ITS Liquid Media Supplement (SIGMA).

Culture of H9C2 rat myoblasts.

H9C2 rat heart‐derived embryonic myocytes (ATCC) were cultured in DMEM (SIGMA), 5% foetal calf serum, penicillin/streptomycin 100 U/ml, 2 mM glutamine, at atmospheric conditions (air plus 5% CO₂).

Neonatal rat ventricular myocyte (NRVM) isolation.

For neonatal cardiomyocytes, hearts of mixed‐sex 2‐ to 3‐day‐old Sprague Dawley rats were dissociated with 0.43 mg/ml collagenase type A (Roche) and 0.5 mg/ml pancreatin (Sigma). Myocyte enrichment was performed by centrifugation through a discontinuous Percoll gradient, and the resultant suspension of myocytes was plated onto gelatin‐coated culture dishes in mix medium containing 80% DMEM High Glucose and 20% M199, supplemented with 10% FBS, 5% HS and 1% antibiotics. After plating, cells were cultivated in mix medium with reduced FBS concentration (5%). Plasmid transfections were performed using Lipofectamine 2000 reagent (Life Technologies).

Mouse embryonic cardiomyocyte isolation and culture.

Under sterile conditions, hearts were removed from mixed‐sex E17.5 embryos and then cut into multiple fragments in cardiomyocyte balanced salt buffer (CBSB; 116 mM NaCl, 20 mM HEPES, 1 mM NaH₂PO₄, 5.365 mM KCl, 831 nM MgSO₄). Heart fragments were then incubated in CBSB with 80 U/ml collagenase type II (Worthington) for 5 min. Allowing fragments to settle, supernatant was removed and centrifuged at 700 × g for 5 min. Remaining fragments were then re‐suspended in cardiomyocyte enzyme solution (CBSB, 80 U/ml collagenase II and 0.25 mg/ml trypsin) for 30 min, with gentle shaking. Fragments were allowed to settle, and then, supernatant containing dissociated cells was removed and centrifuged at 700 × g for 5 min, washed in FBS and stored at 4°C. Remaining fragments were then re‐suspended as before into cardiomyocyte enzyme solution. After centrifugation, the supernatant was discarded and pellet was again re‐suspended in 4°C FBS and placed on ice. Re‐suspension, centrifugation and collection were then repeated until all fragments had been dissolved (7–10 cycles). All FBS suspensions were pooled together and centrifuged at 700 × g for 5 min. The supernatant was then discarded and cells re‐suspended in cardiomyocyte growth medium (Dulbecco's Modified Eagle's Medium (DMEM), supplemented with 17% Medium 199, 5% FBS, 10% horse serum (SIGMA, H0146), 100 μg/ml streptomycin, 100 units/ml penicillin and 2 mM l‐glutamine). Cells were then seeded into a collagen‐coated (1 mg/ml) T75 culture flask (SIGMA) and incubated at 37°C for 2 h. After the incubation, the supernatant, containing cardiomyocytes, was collected from the flask and the adherent fibroblasts discarded. Cardiomyocytes were then cultured in Cardiomyocyte Growth Medium.

Culture and differentiation of AC10 cells.

Cells were seeded at a density of 2.5 × 10⁴/ml on glass coverslips coated with 12.5 μg/ml fibronectin in 0.02% gelatin in DMEM/F‐12 with 12.5% FBS. Once confluent, medium was then replaced by mitogen‐deficient medium, DMEM/F‐12 supplemented with 2% HS and insulin–selenium–transferrin supplement (ITS, Invitrogen), and the cells cultured for a further 2 weeks to allow terminal differentiation.

Transfection of H9C2 myoblasts and rat neonatal cardiomyocytes.

Cells were transiently transfected with a 53BP1‐GFP reporter protein using the plasmid pG‐AcGFP‐53BP1c or either TRF1‐FokI‐D450A or TRF1‐FokI or pBABe‐HA‐ER‐IPpoI plasmids using Lipofectamine 2000 (ThermoFisher) at a ratio of 3 μl Lipofectamine 2000 to 1 μg DNA following the manufacturer's protocol. Transfected cells detected with an anti‐FLAG antibody (SIGMA).

Stress‐induced senescence of cultured cells.

For H9C2 cells, rat neonatal or mouse embryonic CM senescence was induced by 10 Gy X‐ray irradiation. In both cell types, we observed senescence after 10 days using SA‐β‐Gal assay (> 70% positive cells) and absence of proliferation marker Ki‐67. H9C2 senescent or proliferating cells were treated with ABT‐263 (Navitoclax; AdooQ Bioscience, USA), and viability was assessed using a Tali Image‐Based Cytometer (Invitrogen).

EdU incorporation assays.

Cells were incubated in 10 μM EdU in normal growth medium for 24 h. EdU was detected using the Click‐iT® EdU Imaging Kit (Invitrogen), as per manufacturer's protocol.

Thoracic Irradiation.

A TrueBeam linear accelerator (Varian Medical Systems, Palo Alto) was used for the mouse studies. Anesthetised INK‐ATTAC mice (2 months of age) received a single, 20 Gy radiation dose delivered to the thoracic region. The radiation beam was collimated to an area encompassing the mouse lungs and the radiation field positions on the mice were verified using kV‐CBCT and 2D kV imaging of the animals prior to irradiation. The dose rate at the prescription point was 14.8 Gy/min, using 89 cm source to the surface distance. Dose was prescribed to midline in the mice and was confirmed using film and ion chamber dosimetry. All mice prophylactically received 100 mg/ml Baytril antibiotic in their drinking water for 3 weeks post‐irradiation. One month following irradiation, mice were randomised to AP20187 (10 mg/kg), delivered by intraperitoneal injection (treatments twice weekly) or vehicle groups. Body weight was monitored weekly. Mice were euthanised 6 months post‐irradiation exposure using a lethal dose of pentobarbital. Animal experiments involving INK‐ATTAC mice were performed under protocols approved by the Mayo Clinic Institutional Animal Care and Use Committee.

Live‐cell imaging.

For live‐cell time‐lapse microscopy of 53BP1 reporter fluorescence, H9C2 cells were plated on glass coverslip‐bottomed dishes (MatTek), and cells were imaged every 10 min for 10 h as Z‐stacks over 7 μm with a 63× objective (NA = 1.4), using a Zeiss spinning disc confocal microscope with cells incubated at 37°C with humidified 5% CO₂. AcGFP‐53BP1 foci dynamics were analysed using Imaris Software.

Immunohistochemistry.

Deparaffinisation, hydration and antigen retrieval of formalin‐fixed paraffin‐embedded heart tissues were performed as previously described. Sections were incubated with M.O.M mouse IgG blocking reagent (90 μl blocking reagent: 2.5 ml TTBS) for 1 h at room temperature. After 2 × 5 minute PBS washes, sections were incubated with avidin for 15 min, rinsed with PBS, and then incubated with biotin for 15 min at room temperature. Primary antibody was then diluted in M.O.M diluents (7,500 ml TTBS: 600 μl protein concentrate) and incubated overnight at 4°C. After 2 × 5 min PBS washes, sections were incubated in biotinylated‐mouse IgG reagent diluted in blocking solution (1:200) for 30 min at room temperature. After 2 × 5 min PBS washes, endogenous peroxidase activity was blocked by incubating sections in 0.9% H₂O₂ in water for 30 min. After 2 × 5 min PBS washes, sections were incubated in AB complex for 30 min at room temperature. After 3 × 5 min PBS washes, sections were then incubated in NovaRed solution for up to 10 min, rinsed with water, counterstained with haematoxylin for 2 min, washed in PBS and then transferred to ammonia water for 30 s. After a 1‐min wash in water, sections were then dehydrated through 70, 90 and 100% ethanol and then histoclear for 5 min each. Sections were then mounted with DPX.

Primary antibodies used were as follows: mouse monoclonal anti‐4HNE (1:100, MHN; JaICA), mouse monoclonal anti‐8‐OHdG (1:100, MOG; JaICA) and rabbit polyclonal anti‐p21 (1:200, ab7960; Abcam).

Immunofluorescence.

Cells were grown on sterile coverslips, fixed with 2% PFA and permeabilised with PBG‐T (PBS, 0.4% fish‐skin gelatin, 0.5% BSA, 0.5% Triton X‐100). Cells were incubated in primary antibody for 1 h at room temperature (RT), followed by incubation in secondary antibody for 45 min. Primary antibodies: anti‐γ‐H2AX (1:200, #9718; Cell Signaling), anti‐53BP1 (1:200, #4937; Cell Signaling), anti‐53bp1 (1:200, nb100‐305, Novus Biologicals), anti‐α‐actinin (1:100, A7811; SIGMA), anti‐troponin I (1:200, Catalogue, Company), anti‐troponin‐C (1:200, Abcam ab30807), anti‐PCM1 (1:200, ab154142; Abcam), anti‐FLAG (1:1,000, F1804; SIGMA) and anti‐Ki‐67 (1:250 (4 μg/ml), ab15580; Abcam). Secondary antibodies: Alexa Fluor® 488, Alexa Fluor® 594 or Alexa Fluor® 647 (Invitrogen).

Microscopy.

For fluorescence microscopy, a Leica DM5500B wide‐field fluorescence microscope and Zeiss AxioObserver Spinning Disk confocal microscope were used. For super‐resolution STED microscopy, a Leica SP8 confocal (inverted) gSTED 3D super‐resolution microscope was used. For all quantitative immunohistological analysis studies were performed in a blinded manner.

Immuno‐FISH and FISH.

Immuno‐FISH was performed as described in Hewitt et al (2012). Briefly, cells were grown on coverslips and immunocytochemistry performed as described above, with either anti‐γH2AX (1:200, 9718; Cell Signaling) or anti‐53BP1 (1:200, 4937; Cell Signaling). Cells were then fixed with methanol: acetic acid (3:1), dehydrated through an alcohol series, air‐dried and incubated with PNA hybridisation mix [70% deionised formamide (SIGMA), 25 mM MgCl₂, 1 M Tris pH 7.2, 5% blocking reagent (Roche) containing 2.5 μg/ml Cy3‐labelled telomere‐specific (CCCTAA) peptide nucleic acid probe (Panagene)], for 10 min at 80°C in a then for 2 h at RT in the dark humidifier chamber. Cells were washed with 70% formamide in 2× SSC for 10 min, 2× SSC for 10 min and PBS for 10 min. Cells were mounted with ProLong® Gold Antifade Mountant with DAPI (ThermoFisher). For immuno‐FISH in formalin‐fixed paraffin‐embedded tissues, sections were de‐waxed and rehydrated through an alcohol series. Antigen retrieval was via 0.1 M citrate buffer boiling. Sections were incubated in blocked buffer (1:60 Normal Goat Serum in PBS/0.1% BSA) followed by primary antibody at 4°C. Sections were then incubated with a biotinylated secondary antibody (1:200) followed by avidin‐DCS (1:500). Sections were post‐fixed in 4% PFA for 20 min, and FISH was carried out as described above. For centromere‐FISH (SADS detection), a FAM‐labelled, CENPB‐specific (centromere; ATTCGTTGGAAACGGGA) peptide nucleic acid probe (Panagene) was used. Frequency of SADS was assessed by quantification of decondensed/elongated centromeres.

RNA in situ hybridisation.

RNA‐ISH was performed after RNAscope protocol from Advanced Cell Diagnostics, Inc. (ACD). Paraffin sections were deparaffinised with Histoclear, rehydrated in graded ethanol (EtOH), and H₂O₂ was applied for 10 min at RT followed by two washes in H₂O. Sections were placed in hot retrieval reagent and heated for 30 min. After washes in H₂O and 100% EtOH, sections were air‐dried. Sections were treated with protease plus for 30 min at 40°C, washed with H₂O and incubated with target probe (p16, eGFP) for 2 h at 40°C. Afterwards, slides were washed with H₂O followed by incubation with AMP1 (30 min at 40°C) and next washed with wash buffer (WB) and AMP2 (15 min at 40°C), WB and AMP3 (30 min at 40°C), WB and AMP4 (15 min at 40°C), WB and AMP5 (30 min at RT) and WB and, finally, AMP6 (15 min at RT). Finally, RNAscope 2.5 HD Reagent kit‐RED was used for chromogenic labelling. After counterstaining with haematoxylin, sections were mounted and coverslipped. All analysis was performed in a blinded manner.

Crosslinked chromatin immunoprecipitation assay (ChIP).

Three‐month‐old and 30‐month‐old frozen heart tissues (n = 3 per age group) were powdered using dry pulverisation cryoPREP (Covaris). The powder was then re‐suspended into 1% formaldehyde solution in PBS and cells cross‐linked for 5 min at RT, then quenched with glycine buffer at final concentration of 125 mM. The cells were rinsed in cold PBS and harvested into PBS with protease inhibitors and centrifuged for 5 min, 4°C, 1,000 × g. The pellet was re‐suspended in ChIP lysis buffer (1% SDS, 10 mM EDTA, 50 mM Tris–HCl pH 8.1) and incubated for 20 min on ice. Lysate was sonicated to generate average size fragment of 200–700 bp; cell debris was pelleted by centrifugation for 10 min, 4°C, 8,000 × g. The supernatant contained chromatin, which was quantified using OD₂₆₀ on Nanodrop.

Twenty‐five micrograms of chromatin per IP was diluted 1:10 with dilution buffer (1.1% Triton, 1.2 mM EDTA, 16.7 mM Tris–HCl pH 8.1, 167 mM NaCl, with protease inhibitors) and precleared by incubating with 25 μl blocked Staph A membranes (Isorbin) for 15 min. Precleared chromatin was then incubated with 5 μg anti‐γH2AX (phospho S139; Abcam, ab2893) and species‐ and isotype‐matched control ChIP grade IgG (Abcam, ab46540) overnight at 4°C. Immunoprecipitated chromatin was collected with blocked Staph A membranes, which were then sequentially washed with cold buffers; Low Salt Wash Buffer (0.1% SDS, 1% Triton X‐100, 2 mM EDTA, 20 mM Tris–HCl pH 8.0, 150 mM NaCl), High Salt Wash Buffer (0.1% SDS, 1% Triton X‐100, 2 mM EDTA, 20 mM Tris–HCl pH 8.0, 500 mM NaCl), LiCl Wash Buffer (0.25 M LiCl, 1% NP‐40, 1% sodium deoxycholate, 1 mM EDTA, 10 mM Tris–HCl pH 8.0) and two washes in TE Buffer (10 mM Tris pH 8.0, 1 mM EDTA). The immunoprecipitated chromatin was eluted in 500 μl Elution buffer (1% SDS, 100 mM NaHCO₃), cross‐links reversed and DNA obtained by phenol:chloroform extraction and gel purification. Real‐time PCR specific for telomeric repeats was performed as described in Hewitt et al (2012). Briefly, 4 μl of ChIP eluate (bound fraction) was added to a final volume of 13 μl quantitative PCR containing 6.5 μl Jumpstart SYBR master mix (Sigma) and 10 pmol primer mix. Telomere PCR conditions were as follows: 10 s at 95°C followed by 40 cycles of 10 s at 95°C, 30 s at 55°C, and 30 s at 72°C followed by ABI7500 Fast Real‐Time PCR System machine predetermined melt curve analysis (Applied Biosystems). Total input fraction was also collected from chromatin samples. Bound to input (B/I) ratios were determined for each amplicon by taking a 4 μl aliquot of the DNA extracted from the input and bound samples. These were amplified together with a defined amount of genomic DNA on the same plate, using the latter to construct the standard curve. Isotype‐matched irrelevant antibody control (IgG) value was subtracted from the result before plotting the graph. Each PCR was performed in triplicate and results calculated using the following equation: (1/(2A)) × 100.

Senescence‐associated β‐galactosidase activity assay.

SA‐β‐Gal activity assay was performed as described previously (Dimri et al, 1995). For in vitro studies, cells were grown on sterile coverslips, and for in vivo studies, samples were cryo‐embedded and staining performed on 10 μm sections. Samples were fixed with 2% paraformaldehyde in PBS for 5 min, washed with PBS for 5 min and then incubated at 37°C overnight in SA‐β‐Gal solution containing 150 mM sodium chloride, 2 mM magnesium chloride, 40 mM citric acid, 12 mM sodium phosphate dibasic, 5 mM potassium ferricyanide, 5 mM potassium ferrocyanide and 1 mg/ml 5‐bromo‐4‐chloro‐3‐indolyl‐β‐d‐galactosidase (X‐Gal) at pH 5.5. For in vivo staining and quantifications, sections were co‐labelled with anti‐troponin‐C and WGA as above. All analysis was performed in a blinded manner.

TRAP assay.

Heart samples were snap‐frozen in liquid nitrogen immediately after dissection. Using a liquid nitrogen‐cooled pestle and mortar, tissues were ground into a fine powder. Telomerase activity was then determined following the TeloTAGGG Telomerase PCR ELISA kit (Roche).

Assessment of cardiomyocyte hypertrophy.

Hypertrophy was assessed by cross‐sectional area using established methods as described (Shenje et al, 2014). Troponin‐C was used to identify CMs and membranes labelled with wheat germ agglutinin (WGA; Alexa Fluor® 647, W32466, Invitrogen). Only CMs in the left ventricle free wall were analysed. To control for tissue orientation only myocytes that were surrounded by capillaries, all displaying a cross‐sectional orientation were analysed. All analysis was performed in a blinded manner.

Evaluation of cardiomyogenesis in mice.

To allow retrospective quantification of cumulative proliferation and binucleation, occurring the week following navitoclax treatment, mice were injected (intraperitoneal) with EdU (100 μg/g body weight) once daily every 24 h for seven consecutive days (Fig 7A). More than five animals were studied for each treatment group. EdU incorporation was detected using the Click‐iT™ EdU Imaging Kit (ThermoFisher). Sections were also labelled with primary antibody for troponin‐C and the membrane‐specific label WGA. A Leica SP5 laser scanning confocal system was used for the analysis. Analysis was carried out in a blinded fashion similar to our previous studies (Richardson et al, 2015). In brief, myocardial tissue was sectioned transversely in 40 μM sections, with sections separated by intervals of 200 μM, allowing representative analysis throughout the ventricle. Over 80 images were taken, in a random fashion, spanning over eight individual sections per heart. Analysis included the assessment of EdU labelling in > 1,500 cardiomyocytes per heart. Incorporation of EdU in the cardiomyocyte population was quantified based on the detection of EdU labelling in the nucleus of a troponin‐C‐expressing cell. To discriminate proliferation from binucleation (karyokinesis without cytokinesis), the number of EdU‐labelled nuclei within each Edu⁺ cardiomyocyte was assessed using WGA to identify the cell membrane. The incorporation of EdU into a cell with only one nucleus was considered to be a result of proliferation, when two or more EdU‐labelled nuclei were found in the same cardiomyocyte, this was considered to have been incorporated during a multi‐nucleation event. Ki‐67 or Aurora B‐expressing cardiomyocytes were identified as co‐expression of Ki‐67/Aurora B and troponin‐C within a single‐cell membrane (based on WGA labelling). The total percentage of positively expressing cardiomyocytes was calculated as previously described (Richardson et al, 2015), based on the analysis of > 4,000 cardiomyocytes per heart. All quantifications were performed blinded using digital image analysis (ImageJ; U.S. National Institutes of Health; http://rsbweb.nih.gov/ij/).

MR imaging and analysis.

Magnetic resonance images were acquired with a horizontal bore 7.0T Varian microimaging system (Varian Inc., Palo Alto, CA, USA) equipped with a 12‐cm microimaging gradient insert (40 gauss/cm) and analysed as described previously (Redgrave et al, 2017). Percentage change in wall thickening was calculated from the mean of wall thickness at four points in all slices at end systole and end diastole thickness. % change = (EST − EDT)/EDT × 100. All analysis was performed in a blinded manner.

RNA sequencing.

Paired‐end reads were aligned to the mouse genome (mm9) using a splicing‐aware aligner (tophat2). Only unique reads were retained. Reference splice junctions were provided by a reference transcriptome (Ensembl build 67), and novel splicing junctions determined by detecting reads that spanned exons that were not in the reference annotation. True read abundance at each transcript isoform was assessed using HTSeq (Python) before determining differential expression with the tool DESeq2, which models mean‐variance dependence within the sample set. Significance was determined using an FDR‐corrected P‐value ≤ 0.05.

Computational modelling.

Two different models were constructed to examine different hypotheses on how TAF accumulate in cells. The models were encoded in the Systems Biology Markup Language (SBML) modelling standard (Hucka et al, 2003) with the Python SBML shorthand tool (Wilkinson, 2011). Model simulations were carried out in COPASI (Hoops et al, 2006), and the results were analysed and plotted in R using ggplot2 (Wickham, 2009). We used stochastic simulation (Gillespie direct method) in order to be able to account for the variability in the experimental data. The models were deposited in BioModels (Chelliah et al, 2015) and assigned the identifiers MODEL1608250000 and MODEL1608250001.

Cufflinks FPKM determination.

To calculate library normalised read counts for each transcript in every replicate, we used the tool Cufflinks, which returns the proportion of reads per million, which mapped to each gene isoform.

Gene set enrichment analysis (GSEA).

We perform a GSEA by creating a ranked list of the DESeq2 results file, where genes are ranked by their log‐transformed, non‐FDR‐corrected P‐values. This list is then used as an input to the GSEA software, which assesses positive or negative shifts of GO ontology classes in the global distribution of gene expression. This is achieved by the calculation of an enrichment score, which reflects the movement of a particular ontological class to the positive and negative extremes of the distribution of the expression of all genes. Permutation testing then allows for the estimation of significance prior to FDR correction.

Heatmaps.

Heatmaps were created in R using the ggplots package. These heatmaps displaying normalised (row scaled—Z‐score) FPKM gene expression across a series of replicates were clustered using a Pearson correlative clustering approach in the “hclust” R package.

Principal component analysis.

Principal component analysis was performed in R using the prcomp method.