Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact, Peter L.J. de Keizer ( p.dekeizer@erasmusmc.nl ). The following materials: Xpdmice; p16::3MR mice and the peptide FOXO4-DRI are subject to patent applications and may be shared with research organizations for research and educational purposes only under an MTA to be discussed in good faith with the recipient; such MTA may restrict recipient to make any modifications to these materials.

For tissue slice experiments, mice were used for which approval was obtained from the Committee on the Ethics of Animal Experiments of the Erasmus MC, where possible as a left-over from other experiments. Freshly isolated kidneys were sectioned in 200 μM thick slices using a Vibratome (Leica, Eindhoven, the Netherlands). The sections were cultured in Dulbecco’s modified eagle medium with 10% FCS at 37 °C, 5% CO2 on a shaker (60 rpm). Following incubation with shRNA-containing lentiviral particles, or FOXO4-DRI, as indicated, the slices were fixed for 30min in formalin and stored at −80. Subsequently, they were subsectioned to 10 μM slices using a Cryostat, placed on a charged microscopy slide and processed for TUNEL positivity.

The following cell lines were used in this study: IMR90 and WI-38 human fetal lung fibroblasts (female), BJ human foreskin fibroblasts (male), NIH 3T3 mouse fibroblasts, wt and bax/baklittermate Baby Mouse Kidney (BMK) cells, Human Embryonic Kidney (HEK) 293LTV. All cells were obtained from ATCC, except for wt and bax/bakBMK cells which were a kind gift of Dr. Eileen White and have previously been described(). All cells were maintained in high glucose Dulbecco’s Modified Eagle’s Medium (Lonza), supplemented with 10% Fetal Calf Serum, penicillin/streptomycin and 0.05% glutamine. IMR90, BJ, WI-38 cells were kept at 5% CO, 3% Oand used between 28-45 population doublings. The BMK cells were kept at 5% CO, 3% O. The NIH 3T3 and HEK293LTV cells were maintained at 5% CO, ambient O. All cell lines were regularly tested for mycoplasma contamination using MycoAlert Mycoplasma Detection Kit (Lonza) and by assessment of (lack of) DAPI-positive microvesicles under fluorescence microscope. For IR-induced senescence, cells were exposed to 10Gy X- or Gamma rays, and analyzed 10 days later or otherwise indicated; control (proliferating) cells were mock irradiated, meaning they were taken out of the incubator and carried to the irradiator where they were placed outside for the same period of time as the irradiated cells. Senescence was confirmed by SA-β-GAL assay() and/or changes in morphology. For Doxorubicin-induced senescence, the cells were treated twice with 0.1 μM Doxorubicin (Sigma) with a 2d interval and analyzed 7d later, or as otherwise indicated.

This study was performed in strict accordance with all applicable federal and institutional policies. The protocol was approved by the Dutch Animal Ethics Committee. All the mice used in this study were of a C57BL/6J background; either wild-type, Xpd TTD/TTD mutated, expressing p16::3MR, or a combination thereof. The individual strains were backcrossed at least 10 times prior to this study. For the combination Xpd TTD/TTD x p16::3MR the F1 generation was used. The mice were used at the ages indicated in the figures, for Doxorubicin-experiments at 10-40wks of age, for Xpd TTD/TTD versus wt experiments at 26-60 wks of age and for naturally aged mice at 115-130wks of age. All mice were kept in group housing until the start of the experiment after which they were placed in individual cages containing free access to a running wheel. Both sexes were used throughout the study. Where feasible, littermates of the same sex were used. These were randomly assigned to experimental groups.

Method Details

Antibodies and reagents Cip1 (610234), BD Pharmigen: Cytochrome C (556432), R&D systems: IL1α clone 4414, MAB200), human IL6 (AF206NA), Upstate: 53BP1 (05-726), Millipore: FOXO1 (07-702), Santa Cruz: p16Ink4a (JC8; sc56330), PML (N19; sc9862), LMNB1 (M-20; sc-6217), Novacastra: mouse p53 (NCL-p53-CM55). Antibodies against the following proteins were used (See also Key Resource Table ): Cell Signaling: FOXO1 (2880), FOXO4 (9472), Phospho-Ser15 p53 (9286), PUMA (4976), BIM (C3C5), Cleaved Caspase-3 (9661), Sigma: FOXO4 (HPA040232), Tubulin (clone B512; no: T5168), Abcam: BCL2 (Ab7972), IL6 (ab6672), Histone H3 (Ab1791-100), BD Transduction Laboratories: p21(610234), BD Pharmigen: Cytochrome C (556432), R&D systems: IL1α clone 4414, MAB200), human IL6 (AF206NA), Upstate: 53BP1 (05-726), Millipore: FOXO1 (07-702), Santa Cruz: p16(JC8; sc56330), PML (N19; sc9862), LMNB1 (M-20; sc-6217), Novacastra: mouse p53 (NCL-p53-CM55). The following reagents were used: QVD-OPH (BD biosciences), ZVAD-FMK (SelleckChem), Rotenone (Sigma-Aldrich, St. Louis, MO, USA), Doxorubicin (Santa Cruz).

Lentivirus production and shRNAs rd generation production system. In brief, HEK293LTV cells were transfected (Lipofectamine2000) with the packaging/envelope plasmids pRSV-Rev, pMDLg/pRRE and pMD2.G, in combination with a lentiviral transfer plasmid of choice. The next day, the media was refreshed. After 48h, the media was collected for transduction and stored at 4°C until needed. The following shRNA constructs were used (See also de Keizer et al., 2010 de Keizer P.L.

Packer L.M.

Szypowska A.A.

Riedl-Polderman P.E.

van den Broek N.J.

de Bruin A.

Dansen T.B.

Marais R.

Brenkman A.B.

Burgering B.M. Activation of forkhead box O transcription factors by oncogenic BRAF promotes p21cip1-dependent senescence. G12V, we used pLENTI-Puro (670-1)-HRASG12V( Freund et al., 2012 Freund A.

Laberge R.M.

Demaria M.

Campisi J. Lamin B1 loss is a senescence-associated biomarker. Lentiviruses were produced in an MLII-certified lab area, using the 3generation production system. In brief, HEK293LTV cells were transfected (Lipofectamine2000) with the packaging/envelope plasmids pRSV-Rev, pMDLg/pRRE and pMD2.G, in combination with a lentiviral transfer plasmid of choice. The next day, the media was refreshed. After 48h, the media was collected for transduction and stored at 4°C until needed. The following shRNA constructs were used (See also Key Resources Table ): shGFP, shFOXO4-1: TRCN0000039720, Mature sequence: CCAGCTTCAGTCAGCAGTTAT, shFOXO4-2: TRCN0000039721, Mature sequence: CGTCCACGAAGCAGTTCAAAT, shFOXO4(mouse): TRCN0000071560, Mature sequence: GATCTGGATCTTGATATGTAT, shp53-1: TRCN0000003753, Mature sequence: CGGCGCACAGAGGAAGAGAAT and shp53-2: TRCN0000003754, Mature sequence: TCAGACCTATGGAAACTACTT. For transient expression of FOXO1, we transfected cells with pBabe-Puro-FOXO1(). For stable transduction of HRAS, we used pLENTI-Puro (670-1)-HRAS). The day after transduction, the cells were refreshed and 24h later placed on selection in media containing 0.5 μg/ml puromycin. Three days later, the media was refreshed with media containing 1 μg/ml puromycin in which they were kept throughout the assay. After 7d of selection, the cells were used in their respective assays. The shRNA-transduced cells were subsequently exposed to senescence-inducing IR and processed for Cell viability after 6d. For assays on IMR90 cells that were already senescent, treatment occurred as indicated ( Figure S1 C) and the cells were subsequently processed for cell viability or cell density.

FOXO4 D-Retro-Inverso peptide development FOXO4-DRI consists of the following amino acid sequence in D-Isoform: H-ltlrkepaseiaqsileaysqngwanrrsggkrppprrrqrrkkrg-OH. MW: 5358.2 It was manufactured by Pepscan (Lelystad, the Netherlands) at > 95% purity and stored at −20°C in 1mg powder aliquots until used to avoid freeze-thawing artifacts. For in vitro experiments FOXO4-DRI was dissolved in PBS to generate a 2mM stock. For in vivo use, FOXO4-DRI was dissolved in PBS to generate a 5mg/ml stock solution, which was kept on ice until injection. Before injection the solution was brought to room temperature.

Total RNA isolation and mRNA sequencing sample preparation The total RNA isolation and sequencing library preparation from IR-induced senescent and proliferating IMR90 cells was performed as follows: total RNA was isolated using Qiazol Lysis Reagent (QIAGEN) and purified with the miRNeasy kit (QIAGEN). The integrity (scores > 9.0) of the RNA was determined on the Agilent 2100 Bioanalyzer (Agilent). Total RNA enrichment for sequencing poly(A) RNAs was performed with the TruSeq mRNA sample preparation kit (Illumina). 1μg of total RNA for each sample was used for poly(A) RNA selection using magnetic beads coated with poly-dT, followed by thermal fragmentation. The fragmented poly(A) RNA enriched samples were subjected to cDNA synthesis using Illumina TruSeq preparation kit. cDNA was synthesized by reverse transcriptase (Super-Script II) using poly-dT and random hexamer primers. The cDNA fragments were then blunt-ended through an end-repair reaction, followed by dA-tailing. Subsequently, specific double-stranded bar-coded adapters were ligated and library amplification for 15 cycles was performed. The pooled cDNA library consisted of equal concentration bar-coded samples. The pooled library was sequenced in one lane, 36 bp single read on the HiSeq2500 (Illumina). The analysis of the sequencing dataset was performed by Total RNA analysis pipeline (TRAP).

Total RNA analysis pipeline (TRAP) Reads were aligned to the human hg19 reference genome using the NARWHAL automation software. Exonic reads were summed per transcript. A specific transcript was considered expressed, when a minimum number of reads, i.e., 5 reads per million, could be aligned to a transcript. Pathway analysis was performed with Ingenuity Pathway Analysis Software (IPA; Version build 242990).

Production and purification of recombinant proteins The constructs corresponding to human FOXO4 (residues 86 – 206) and human p53 (residues 1 – 312), were purchased from Genscript in a pUC cloning vector. The DNA sequence was codon optimized for protein production in bacterial cells and flanked by NcoI and BamHI restriction sites. The coding region was cloned into a modified pETM-11 bacterial expression vector (EMBL Heidelberg) which was derived from a pET-24d(+) vector (Novagen) by insertion of a tobacco etch virus (TEV) protease cleavage site following a N-terminal hexa-histidine and protein A tag. The genes were amplified by PCR using T4 primers (New England Biolabs). The resulting PCR products and pETM-11 were double digested with NcoI and BamHI enzymes (New England Biolabs) before ligation. The construct was verified by sequencing. The human p531-312, and FOXO486-206 DNA sequences were codon optimized for protein production in bacterial cells and flanked by NcoI and BamHI restriction sites. The coding region was cloned into a modified pETM-11 bacterial expression vector which was derived from a pET-24d(+) vector (Novagen) by insertion of a tobacco etch virus (TEV) protease cleavage site following an N-terminal hexa-histidine and protein A tag. Unlabeled and uniformly (15N) labeled protein was produced in freshly transformed E. coli BL-21 (DE3) cells. A single colony was inoculated in Luria-Bertani medium (20 ml) with kanamycin (25 mg l-1) and cultured at 37°C overnight. From this, an aliquot (1 ml) was added to either 1 l unlabeled Luria-Bertani medium or 1 l 15N labeled M9 minimal medium (100 mM KH 2 PO 4 , 50 mM K 2 HPO 4 , 60 mM Na 2 HPO 4 , 14 mM K 2 SO 4 , 5 mM MgCl 2 ; pH 7.2 adjusted with HCl and NaOH with 0.1 dilution of trace element solution (41 mM CaCl 2 , 22 mM FeSO 4 , 6 mM MnCl 2 , 3 mM CoCl 2 , 1 mM ZnSO 4 , 0.1 mM CuCl 2 , 0.2 mM (NH 4 ) 6 Mo 7 O 24 , 17 mM EDTA) with kanamycin (25 mg l-1) in which 15N-NH 4 Cl (2 g l-1) was the only source of nitrogen for NMR isotope labeling purposes, respectively (Cambridge Isotope Laboratories, Inc). The culture was incubated at 37°C and shaken at 200 rpm until the OD600 reached 0.8. Protein expression was induced with 1 mM β-D-1-thiogalactopyranoside (IPTG) at 18°C. The cells were pelleted after 20 hr by centrifugation using a Fiberlite F9-6x1000 rotor in a Sorvall LYNX 6000 Superspeed centrifuge at 2,000 g for 20 min, re-suspended in 40 mL lysis buffer containing 50 mM Tris, pH 7.5, 150 mM NaCl, 20 mM imidazole, 20% glycerol, 2 mM TCEP, 250 units of Benzonase® Nuclease and bacterial Protease Inhibitor Cocktail (Amresco; AEBSF, E-64, Bestatin, EDTA and Pepstatin) and were subsequently lysed by sonication. Lysates were cleared by centrifugation for 45 min at 4°C (12 000 rpm; Sorvall Lynx 6 000) and filtration (0.45 μm cellulose acetate syringe filters). Proteins were purified using Ni-NTA resins for immobilized metal affinity chromatography. Lysate was applied on the column and contaminants were removed using Wash buffer 1 (50 mM Tris, pH 7.5, 150 mM NaCl, 20 mM imidazole and 2 mM TCEP) and Wash buffer 2 (50 mM Tris, pH 7.5, 1 M NaCl, 20 mM imidazole and 2 mM TCEP). Protein was eluted (50 mM Tris, pH 7.5, 1 M NaCl, 500 mM imidazole and 2 mM TCEP) and the His6-Protein A tag was cleaved overnight at 4°C using 2 (w/w) % of 1 mg/ml recombinant His-tagged TEV protease. The protein solution was buffer exchanged to Wash buffer 1 using HiPrep 26/10 Desalting column on an ÄKTA Pure system (GE Healthcare) at room temperature. Cleaved protein was repurified from the cleaved fusion tag and TEV protease by loading on a Ni-NTA resin column and 5 mL of the flow through was applied to a size-exclusion column (HiLoad Sepharose 16/600 75 pg, GE Healthcare) on an ÄKTA Pure system (GE Healthcare) at RT. The corresponding running buffer was composed of 50 mM sodium phosphate, pH 6.5, 150 mM NaCl and 1 mM DTT.

NMR spectroscopy Samples for NMR measurements contained 300 μM 15N labeled FOXO486-206 protein in 50 mM sodium phosphate, pH 6.5, 150 mM NaCl and 1 mM DTT with 10% 2H 2 O added for the lock signal. For NMR titrations, 0.2, 0.4, 0.8 and 1.0 stochiometric equivalents of p531-312 (corresponding to 60, 120, 240 or 300 μM, respectively) and 1.0 and 2.0 stochiometric equivalents of FOXO4-DRI (corresponding to 300 or 600 μM, respectively) were added. 1H-15N HSQC NMR spectra were recorded at 298 K on a 700 MHz Bruker NMR spectrometer. All spectra were recorded with an interscan delay of 1.0 s, spectral widths of 15.9/30 ppm, centered at 4.7/118.0 ppm in 1H/15N, with 1,024 and 256 points, respectively, and 16 scans per increment. NMR chemical shift assignments of FOXO486-206 were obtained from HNCACB and HNCA spectra using 500 μM of uniformly 15N,13C labeled protein in the aforementioned buffer at 298K on a 900 MHz Bruker Avance III NMR spectrometer equipped with a TCI cryoprobe.

Immunofluorescence and Structured Illumination Microscopy Cells (typically 20,000) were grown on coverslips and after the indicated treatment fixed with formalin. Subsequently, cells were washed in Tris Buffered Saline (TBS) and permeabilized for 2-5 min in 2% Triton X-100 in TBS, depending on the antibody. Especially, the FOXO4 antibody (Cell Signaling) seemed to work better with shorter permeabilization times. To reduce background staining, the cells were quenched for 10min with 50nM glycine in TBS and blocked for 30min with 5% Normal Horse Serum (NHS) or Normal Goat Serum (NGS) in 0,2% gelatin-TBS solution, depending on the isotype of the secondary antibody. Subsequently, 30μl droplets containing primary antibody dilutions were placed on parafilm in a dark moisture chamber. The coverslips were placed facing the droplets and incubated overnight at 4°C. The next day, the coverslips were lifted by adding a small volume (∼200μl) of TBS-Gelatine under the coverslip and transferred back to the 24-well plate. After washing 3x 20min with 1ml 0,2% gelatin-TBS secondary antibody incubation occurred as described for the primary antibody and the coverslips were incubated for one hour at room temperature. Following 3x 10min washes with 1ml 0,2% TBS-Gelatin and 1 wash with regular TBS the slides were mounted using soft set mounting medium with DAPI (Vectashield) and sealed with nail polish. Structured Illumination Microscopy was performed using a Zeiss Elyra PS1 microscope using a 63x 1.4 (n.a.) plan apo chromate oil immersion lens and 5 phases and 5 rotations of the illumination pattern. Intensity plots of individual pixels taken from a straight line in indicated immunofluorescence images were generated by twin slicer analysis using Huygens Professional 4.0 software (SVI, the Netherlands). Images were cropped and processed in Adobe Photoshop. When comparisons were made between images of the same experiment, all levels were adjusted equally and the ratio between the levels was not altered.

Quantitative real-time PCR FOXO1 ( NM_002015.3 ): Fwd: tggttttagaaacccaagttcc, Rev: ttggcaccaagttcagttaca. UPL75

FOXO3 ( NM_001455.3 ): Fwd: cagtagggcctgtgatttcc, Rev: cagcagaccaacactgttcac. UPL73

FOXO4 ( NM_005938.2 ): Fwd: acgagtggatggtccgtact, Rev: gtggcggatcgagttcttc. UPL18

P21Cip1 (CDKN1A; NM_000389.3 ): Fwd: cgaagtcagttccttgtggag, Rev: catgggttctgacggacat. UPL82

ETS2 ( NM_005239.4 ): Fwd: cagcgtcacctactgctctg, Rev: agtcgtggtctttgggagtc. UPL27

Tubulin ( NM_006009.2 ): Fwd: cttcgtctccgccatcag, Rev: ttgccaatctggacacca. UPL58 mRNA was extracted using the Cells-to-Ct kit (Ambion). QPCR was performed using the Universal Probe Library system (Roche) with the following primer/probe combinations (5′-3′), also listed in Table S1

Immunoblotting The dishes were washed 2x with ice-cold PBS, lysed in 1x Laemli sample buffer, and subjected to standard SDS-PAGE using separate 4%–12% Bis-Tris gels, after which the proteins were overnight transferred at 4°C to polyvinylidene fluoride membranes. The membranes were blocked using 2% BSA in Tris-buffered saline, 0.05% TWEEN (TBS-T) for 60min and incubated overnight at 4°C with the indicated primary antibodies. Following at least two 20min washing steps with TBS-T, the membranes were incubated with secondary antibodies for 60min. Following at least two 15min washing steps the membranes were developed with Enhanced Chemical Luminescence (Perkin Elmer). Images were cropped and equally processed in Adobe Photoshop.

Cell viability assays The cells were plated in triplicate in 96-well plates (typically 7000 senescent and 2000 non-senescent cells). Unless otherwise indicated, cell viability was assessed 6d after plating, using the AQueousOne Solution Cell Proliferation Assay (Promega). 10 ul of CellTiter AQueousOne Solution in 100ul fresh culture medium was added to the wells before a 1-3 hr incubation at 37°C. Absorbance was measured at 490nm at a GloMax 96 well plate reader (Promega). A Mock-treated and a Puromycin (10 μg/ml)-treated condition were used to set the maximal and minimal viability values, respectively, to which the experimental values were normalized.

Cell density assays Stably transduced and Puromycin-selected IMR90 cells were plated in 24-well plates in triplicate. After 3d, cells were fixed in methanol and stained with 0.5% crystal violet in 25% methanol. The plates were dried, and cell density was quantified by destaining in 10% acetic acid and measuring absorbance of the solution at 560 nm.

Apoptosis assays To assess apoptosis, two separate assays were used. TUNEL staining was performed by permeabilizing cells fixed on coverslips for 2min with 0.1% Triton X-100 in 0.1% sodium citrate, followed by labeling with 10% TUNEL enzyme versus label solution for 45min (Roche). Objective analysis of the percentage of TUNEL-positive cells was performed using CellProfiler software v2.3 by scoring the # of TUNEL positive objects filtered over DAPI-positive objects (Nuclei). Only TUNEL positive objects were considered that were also DAPI positive. For the Cytochrome-C release assay, cells were seeded on coverslips in a 24-well plate and incubated for 5d total with the pan-caspase-inhibitor QVD-OPH (20 μM in 500μl). The media was refreshed on day 3. Subsequently, the cells were processed for Cytochrome-C positivity by immunofluorescence. We measured the percentage of cells that showed a mitochondrial release of mitochondrial Cytochrome-C, identified either as diffuse staining, or complete absence.

Real-time cell density assay Real-time cell density was measured using an xCELLigence detection system (ACEA Biosciences). Prior to the measurement, 50 μL DMEM 10% FCS was added to each well of an E-plate view 16 (Roche) to establish background signal. Non-senescent (2000 cells per well) and senescent (5000 cells/well) IMR90 fibroblasts were then plated in 150 μl medium. 16h later the E-plate was placed in the xCELLigence reader and the cell density was recorded every 30min. The cells were treated with 25uM FOXO4 peptide 8h after starting the measurements. Measurements continued for the indicated intervals.

Real-time imaging of Caspase-3/7 activation Cells were plated in 4-well Poly-L-Lysine coated glass bottom 35mm dishes (D141410; Matsumi, Japan) and incubated with NucView488 Caspase-3 (4440; Essen Bioscience). FOXO4-DRI or PBS was added and the cells were transferred to a Heat and CO 2 -controlled incubator, attached to a LSM510 confocal microscope (Zeiss). 8h after addition, real-time imaging was initiated and every 30min a grid of 3x3 pictures was recorded. The imaging continued for another 6 days and the images were concatenated using Zen imaging software (Zeiss; See Mov3+4).

Genotyping of mice For p16::3MR-1: p16::3MR1: 5′-AACGCAAACGCATGATCACTG-3′ and

p16::3MR-2: 5′-TCAGGGATGATGCATCTAGC-3′. Positive animals show a band at 202bp.

For XpdTTD/TTD: p145: CCCGGCTAGAGTATCTGC, p184: GCCGGAATACGGGGCCA and

pβrev:TCTATGGTTAAGTTCATGTCATAGGAAGGGGAGAA. For PCR genotyping the following primers were used (See also Table S1 ):

Design of mouse experiments α/2 +Z β )2 ∗ σ2/Δ2, with a power (1-β) of 80% and a significance (α) of 0.05. Prior to the study, pilot experiments were performed to determine the differences in plasma [Urea] of 26wk old wt versus XpdTTD/TTD mice. This led to a Δ and σ such that a sample size of 7 mice per group were estimated to be required to see differences in such experiments. This is shown in For the calculation of the estimated sample size a power analysis was performed according to the formula: n = 2(Z+Z/Δ, with a power (1-β) of 80% and a significance (α) of 0.05. Prior to the study, pilot experiments were performed to determine the differences in plasma [Urea] of 26wk old wt versus Xpdmice. This led to a Δ and σ such that a sample size of 7 mice per group were estimated to be required to see differences in such experiments. This is shown in Figure 6 K. Later, is was found that sample sizes could be reduced when using mice of older age. This was applied in Figure 6 L and Figure 7 . For other assays, no information on Δ and σ was available and similar, or less stringent, sample sizes were deemed necessary. We only mice included mice that were of sufficient body weight at the start of the experiment, typically at least 80% of the average littermate weight of the same sex and genotype.

Assessment of running wheel activity st treatment) the average running wheel activity over 4 days was scored. The ratio of mice of both sexes from four independent experiments was calculated and the % activity plotted in For assessment of running wheel behavior, we only included mice which at baseline ran at least 0.1km/day. Running wheel activity was continuously measured and plotted in km/day. The mice were placed in running wheel cages with ample time to adjust and get trained in using the wheel. After withdrawal of a blood sample, the mice were allowed to recover for at least 1 day and the average running wheel activity over the next two days was taken as baseline value. The mice were subsequently treated with FOXO4-DRI, or PBS (Mock) and at t = 21d after baseline (t = 18d after 1treatment) the average running wheel activity over 4 days was scored. The ratio of mice of both sexes from four independent experiments was calculated and the % activity plotted in Figure 5 I. Note that in some cases blood samples were taken at t = 9 after treatment causing a temporary dip in activity.

Fur density analysis TTD/TTD mice show reduced fur density ( de Boer et al., 2002 de Boer J.

Andressoo J.O.

de Wit J.

Huijmans J.

Beems R.B.

van Steeg H.

Weeda G.

van der Horst G.T.

van Leeuwen W.

Themmen A.P.

et al. Premature aging in mice deficient in DNA repair and transcription. de Boer et al., 1998 de Boer J.

de Wit J.

van Steeg H.

Berg R.J.

Morreau H.

Visser P.

Lehmann A.R.

Duran M.

Hoeijmakers J.H.

Weeda G. A mouse model for the basal transcription/DNA repair syndrome trichothiodystrophy. Xpdmice show reduced fur density (). To score any changes that might occur over time, the phenotype was ranged from 0-4 where 0 was very patched and 4 was wild-type. Each mouse was scored before and after the experiment as indicated in Figure 5 C and Figure S5 A. The final score was determined as the ratio (final-baseline)/baseline and the % change was subsequently plotted. Following several initial pilot observations, the experiment shown in Figure 5 C contains mice from two independent cohorts. For naturally aged mice ( Figure 7 D), only males were included, since we did not observe significant hair loss in females. In these cohorts, 80% showed (varying degrees of) loss of hair at the beginning (115+wks), or developed it over the course of the experiment.

Infrared-measurements of abdominal temperature TTD/TTD mice, there appeared to be a difference in surface temperature. This we reasoned to be caused by changes in fur density. To further quantify such changes, the abdominal temperature was subsequently measured using an infrared thermometer. Even though the variation in individual measurements per animal was relatively large, XpdTTD/TTD mice showed a significantly higher average infrared-measured abdominal temperature in general (See also From handling wt versus Xpdmice, there appeared to be a difference in surface temperature. This we reasoned to be caused by changes in fur density. To further quantify such changes, the abdominal temperature was subsequently measured using an infrared thermometer. Even though the variation in individual measurements per animal was relatively large, Xpdmice showed a significantly higher average infrared-measured abdominal temperature in general (See also Figure 5 E). There were also mice with a relatively normal temperature. To assess the effect of FOXO4-DRI versus PBS therefore only mice with a baseline temperature > 34 degrees were included as the window of visualizing any changes would otherwise be too small.

Immunohistochemistry For immunohistochemistry, paraffin sections of liver and intestine specimens were deparaffinized, rehydrated in decreasing concentrations of ethanol, treated for 10 min with 3% H 2 O 2 to quench endogenous peroxidase activity and heated to 100°C for 1 hr in 10 mM sodium citrate buffer, pH 6, for antigen retrieval. Subsequently the tissues were processed as for immunofluorescence. Paraffin embedded heart tissue was serially sectioned into 5 μm slices, deparaffinized and rehydrated before Hematoxylin-Eosin (HE) staining or Picro Sirius Red staining was performed. For the HE staining, the sections were stained in Gills Hematoxylin (Sigma) for 4 min and 30 s in Eosin-Y solution. To stain for collagen, sections were incubated for 60 min in Picro Sirius Red solution (1g/L in picric acid) and briefly rinsed twice in 0.05% acetic acid.

Bioluminescence For in vivo luminescence, mice were injected i.p. with 15 mg of Xenolight RediJect Coelentarazine (Caliper). 20 min later, the mice were anesthetized (2%–4% isoflurane) and placed in a dorsal position during imaging. Bioluminescence was measured with a Xenogen IVIS-200 Optical in vivo imaging System (Caliper Life Sciences; 5 min exposure). Photon flux was quantified within a circular region of interest (ROI) encompassing the site of substrate injection and the total radiance was corrected for time and surface area measured.

Plasma values as measure for tissue function On the indicated time points, whole blood samples were collected in a Microvette with Lithium Heparin (Sarstedt) for plasma separation and spun for 10min at 4.6 x g. The (clear) supernatant was transferred into regular 1.5ml tubes and spun again for 5min at 4.6 x g. The supernatants were transferred again into 1.5ml tubes, snap frozen in liquid N 2 and stored at −80°C. [AST] was measured using an AST Activity Assay Kit (Sigma). The samples were incubated with 100μl reaction mix in a 96 well plate and placed at 37°C. The absorbance at 450nm was determined after 2 min for baseline analysis and after 40 min for a final analysis. [Urea] was measured using a QuantiChrom Urea Assay Kit (Gentaur). The samples were incubated in 200μl reaction mix for 10 min at room temperature before absorbance was measured at 520nm. [Creatinine] was measured using Creatinine Assay Kit (Sigma). Samples were incubated with 50μl reaction mix at 37°C for 60 min and the absorbance was measured at 570nm. Ratios comparing plasma values after treatment compared to baseline were determined and plotted as % over baseline in scatterplots.