p53 helps fix DNA and reduces DNA fragments and inflammation in aging cells

This study was approved by the Institutional Animal Care and Use Committee at Sanford Burnham Prebys MDI (AUF 22-005). C57BL6 mice were purchased from Jackson Labs and Charles River and group housed at 21–24 °C, 30–70% humidity, and a 12 h light/dark cycle under specific pathogen free conditions with ad libitum access to water and food (Teklad 2018). Cohort 1 used 21 month-old female and 24-month old male C57BL6J mice and 4 month-old male and female C57BL6N mice. Cohort 2 used 22 month-old and 6 month-old female C57BL6J mice. Mice were treated with 10 mg/kg HDM201 (Novartis) suspended in phosphate-buffered methylcellulose according to the manufacturer's instructions (0.5% methylcellulose in phosphate buffer, pH6.8, 50 mM) by daily oral gavage in the morning. Mice were monitored daily and weighed every few days during treatment. After 14 days of treatment, mice were euthanized by CO2 asphyxiation in the morning, and tissues were fixed in 10% neutral buffered formalin (Epredia 9400-1) or flash frozen in liquid nitrogen, either whole or in OCT (Tissue-Tek 4583). Whole blood was counted using a hematology analyzer (Abaxis VetScan HM5).

IMR90 primary human fibroblasts were purchased from the American Type Culture Collection (CCL-186) and grown at 37 °C, 3.5% O2, 5% CO2, in Dulbecco's modified Eagle's medium (Gibco 10313-121) with 10% FBS (Corning 35-0-11-CV), 1% penicillin/streptomycin (Gibco 15140-122) and 2 mM glutamine (Gibco 25030-081). Cells were checked routinely for mycoplasma contamination and tested negative. IR senescence was induced by 20 Gray x-ray irradiation of 20-30% confluent cells. Cells were split after returning to confluence in 3 days, then treated with MDM2 inhibitors, RG7388 (Selleckchem) and HDM201 (Novartis), starting day 4 after irradiation or day 5 after irradiation in experiments that also included siRNA treatment, unless otherwise noted. Unless otherwise indicated, cells were collected 10 or 11 days after irradiation. For longer-term treatments, drugs were replaced by media change every 3 days. RG7388 was used at 12.5 nM, or 100 nM (Fig. 1B; Fig. S2H; Fig. 2G; Fig. S3E, F), and HDM201 was used at 25 nM. IMR90 cells exogenously expressing Parkin and the method for mitochondria ablation are previously described (Vizioli) and 10 μM KU55933 was used for ATM inhibition experiments. For etoposide-induced senescence, 80% confluent IMR90 cells were treated with 50 mM etoposide for 48 h, RG7388 was added on day 4 after initiation of senescence, and cells were collected on day 7. 293 T cells were purchased from the American Type Culture Collection (CRL-3216). I9A human fibroblast cells⁴⁰ were a gift from Dr. Vera Gorbunova. No commonly misidentified cell lines were used in this study.

pLV-hPGK-HA-53BP1-puro and pLV-EF1A-FLAG-p53-puro constructs were generated by Vectorbuilder. Mutations to 53BP1 are described in Fig.. pLV-TetOne-eBFP2-I-SceI-Puro was a gift from Dr. Vera Gorbunova. S1F

For 53BP1 exogenous expression, lentivirus was generated from 293 T cells transfected with expression vector, VSVG envelope vector, and pMD2.G packaging vector in lipofectamine 2000 (Invitrogen 52887). Virus was collected over 3 days, then concentrated by ultracentrifugation. For p53 exogenous expression, concentrated lentivirus was generated by the SBP viral vector core facility using a proprietary protocol. Concentrated virus was titrated to the minimum amount required for > 90% cell viability after puromycin selection (1 μg/mL for 72 h). Infections were overnight (16 h), in the presence of 8 μg/mL polybrene (Millipore TR-1003-G).

Senescent cells were transfected 4 days after irradiation with 100 nM siRNA (Dharmacon siGENOME) in 0.8% Dharmafect reagent (Dharmacon) according to manufacturer's recommendations. Experiments used a pool of four siRNAs per gene unless otherwise noted. Cells were changed into regular media 18–20 h later, except sip21 experiments.

The following primary antibodies were used: 53BP1 (Cell Signaling Technology Cat#4937, RRID:AB_10694558), ATM (D2E2) (Cell Signaling Technology Cat#2873, RRID:AB_2062659), CENPA (Thermo Fisher Scientific Cat#MA1-20832, RRID:AB_2078763), Cyclin A (Santa Cruz Biotechnology Cat#sc-271682, RRID:AB_10709300), ANTI-FLAG M2 (Sigma-Aldrich Cat#F3165-2MG, RRID:AB_259529), Phospho-Histone H2A.X (Ser139) (Millipore Cat#05-636, RRID:AB_309864, Active Motif Cat#39117, RRID:AB_2793161), HA-probe (F-7) (Santa Cruz Biotechnology Cat#sc-7392, RRID:AB_627809), IgG control (Vector Laboratories Cat#I-1000, RRID:AB_2336355, Vector Laboratories Cat#I-2000, RRID:AB_2336354), IL8 (Abcam Cat#ab18672, RRID:AB_444617), MDM2 (Cell Signaling Technology Cat#51541, RRID:AB_2936381), p21 (Santa Cruz Biotechnology Cat#sc-817, RRID:AB_628072), p53 (Santa Cruz Biotechnology Cat#sc-126, RRID:AB_628082, Leica Biosystems Cat#NCL-p53-CM5p, RRID:AB_563933), Phospho-p53 (Ser15) (Cell Signaling Technology Cat#9284S, RRID:AB_331464), Phospho-ATM (Ser1981) (Abcam Cat#ab81292, RRID:AB_1640207), TOMM20 (Abcam Cat#ab56783, RRID:AB_945896). The following secondary antibodies were used: Goat anti-Mouse IgG, IgM (H + L) HRP (Thermo Fisher Scientific Cat#31446, RRID:AB_228318), Goat anti-Rabbit IgG, (H + L) HRP (Millipore Cat#AP307P, RRID:AB_92641), Goat anti-Mouse IgG (H + L), Alexa Fluor^TM 594 (Thermo Fisher Scientific Cat#A11032, RRID:AB_2534091), Goat anti-Rabbit IgG (H + L), Alexa Fluor^TM 488 (Thermo Fisher Scientific Cat#A11008, RRID:AB_143165). Antibodies used for flow cytometry are listed in Supplementary Data 3.

For cells, blotting was done using standard approaches⁶⁸. Briefly, cells were lysed in modified RIPA buffer (50 mM Tris-Cl pH 7.5, 0.25% sodium deoxycholate, 150 mM NaCl, 10 mM EDTA, 0.1% SDS, 1% Igepal, 1x dual protease and phosphatase inhibitor (Thermo 1861281)) and lysates were cleared by > 20000 g centrifugation. Protein was quantified by BCA assay (Pierce 23225), mixed with sample buffer, run on precast gels (Biorad), and transferred either with a Turbo semidry system (Biorad), or for 53BP1 experiments wet transfer in tris-glycine buffer (Biorad) with 5% methanol. Membranes were blocked in milk and imaged by ECL (Thermo 34095, Biorad Chemidoc). For mouse liver tissue, samples were lysed in modified RIPA buffer using a Bertin Technologies Precellys tissue disruptor. Full-length blots with size markers are supplied in the Source Data file.

Protein G Dynabead-antibody complexes were prepared as previously described²². Cells were washed 4 times in PBS, scraped in EBC500 (50 mM Tris-Cl pH 8, 500 mM NaCl, 0.5% Igepal, 2.5 mM MgCl2) with benzonase (250 U/mL), and lysed by rotating 30 minutes at 4 °C. Protein was quantified by BCA assay. Immunoprecipitations were run overnight at 4 °C, washed 7 times in NETN (20 mM Tris-Cl pH 8, 100 mM NaCl, 1 mM EDTA, 0.5% Igepal), and eluted in sample buffer.

Cells were plated on PhenoPlate^TM 96-well microplates (PerkinElmer), stained as described previously²², and imaged on a Nikon T2 microscope with automated image capture. Images were analyzed in NIS Elements AR v5.21.03 using dark background subtraction, thresholding, size exclusion, and automated partitioning to identify features. Measurements indicate total number of features divided by total number of nuclei, except in Fig. 5, where total mitochondrial or nuclear γH2A.X staining area is divided by total number of nuclei.

Staining was done as previously described²¹.

This assay was carried out following manufacturer's instruction for the neutral comet SCGE assay (Enzo, #ADI-900-166). Slides were placed flat in the dark at 4 C in the dark for 30 minutes. Slides were immersed in pre-chilled lysis solution (2.5 M NaCl, 100 mM EDTA pH10, 10 mM Tris Base, 1% sodium lauryl sarcosinate, 1% Triton x-100, Catalog No.4250-050-01) for 45 minutes. Electrophoresis was performed in TAE buffer (40 mM Tris base, 20 mM acetic acid, 1 mM EDTA disodium salt dihydrate) at 30 V for 15 minutes at room temperature. Automated comet analysis was performed using an open-source tool in ImageJ (Gyori et al 2014).

qPCR: Cells were lysed in Trizol (Ambion 15596026) and RNA was isolated using either a commercial kit (53BP1 and MDM2i RNAseq experiments; Direct-zol RNA Miniprep Kits, Zymo Research Cat#R2050) or with chloroform according to the manufacturer's protocol. RNA was converted to cDNA (RevertAid Reverse Transcriptase, Thermo Fisher Scientific Cat#EP0441, Ribolock RNase Inhibitor, Thermo Fisher Scientific Cat#EO0381, 5x reaction buffer for RT, Thermo Fisher) and gene expression quantified by a standard SYBR-based approach (PowerUp^TM SYBR^TM Green Master Mix for qPCR, Applied Biosystems Cat#A25741, QuanStudio^TM 6 Flex Real-Time PCR System, 384-well, Applied Biosystems Cat#4485691).

RNA was quantified by bioanalyzer and library preps were made by the SBP genomics core. Sequencing was done by the SBP genomics core or at UCSD Institute for Genomic Medicine. For analysis, raw fastq files were aligned to hg19 (53BP1 OE RNAseq) or hg38 (MDM2i in cell culture RNAseq), or mm10 (MDM2i in mice RNAseq) using STAR⁶⁹ 2-pass pipeline. Reads were filtered, sorted and indexed by SAMtools⁷⁰. FPKM were generated using CuffLinks⁷¹ for downstream visualization. Genome tracks (bigWig files) were obtained by Deeptools⁷². Raw read counts were obtained by HTSeq⁷³ for differential analysis. Differentially expressed genes were obtained by DESeq2⁷⁴. KEGG gene ontology was run using WebGestalt⁷⁵, gene lists were compared using Venny2.1(https://bioinfogp.cnb.csic.es/tools/venny/index.html↗), and heatmaps were generated using Morpheus (https://software.broadinstitute.org/Morpheus↗).

Cells were trypsinized, washed in PBS, and resuspended at 20x cell pellet volume in cold nuclear isolation buffer A with digitonin (50 nM HEPES pH 7.3, 150 mM NaCl, 1x dual protease and phosphatase inhibitor, 25 μg/mL digitonin) by pipetting. The suspensions were rotated at 4 °C for 30 minutes, then centrifuged at 500 g for 5 minutes at 4 °C. Nuclear pellets were washed twice with cold NIB-250 buffer (250 mM sucrose, 15 mM Tris-Cl pH 7.5, 60 mM KCl, 15 mM NaCl, 5 mM MgCl2, 1 mM CaCl2) and resuspended in sorting buffer (DPBS with 2% FBS, 0.5 mM spermidine, 500 ng/mL DAPI). Nuclei were sorted by FACSAriaII with a 100um nozzle (see also Fig. S7) into individual strip tubes, and libraries were generated by LIANTI⁴³ or PicoPLEX Gold (Takara Bio). Libraries were sequenced by NOVAseq (Illumina). For analysis, the first 14 bases of both R1 and R2 reads were trimmed using CutAdapt⁷⁶. Trimmed reads were then aligned to hg38 using Bowtie2⁷⁷. The sam files were transferred to bam files, then sorted and indexed using SAMtools⁷⁰. Duplicates were removed using picard tools (https://broadinstitute.github.io/picard/↗) MarkDuplicates function. Copy number variation profiles were obtained via Ginkgo⁷⁸.

I9A cells were irradiated at 20 Gy, split 1:2 three days after irradiation, then on the next day infected with lentivirus containing I-SceI and treated simultaneously with 1 μg/mL doxycycline and 100 nM RG7388 or DMSO. 16 h later, virus was removed and treatments were refreshed. Cells were collected on day seven after irradiation and analyzed by flow cytometry using a BD LSRFortessa Cell Analyzer.

IMR90 cells were pretreated for 30 minutes with 12.5 nM RG7388 or DMSO, then treated in reverse with 100 μM cycloheximide for 1 or 3 h. The 0 h control was treated with DMSO for 3 h.

Tissues were dissected and placed on ice-cold RPMI supplemented with 10% FBS. Single-cell suspensions of immune cells from the liver were obtained by mechanical disaggregation through a 70 µm cell strainer (VWR) and washed through with 10% FBS in RPMI. Liver samples were spun at 60 r.c.f. and 4°C for 2 min with no brake to pellet hepatocytes before percoll (Cytiva) centrifugation. The supernatant was collected, spun at 420 r.c.f. and 4°C for 4 min. The pellet was resuspended with 40% Percoll (Cytiva) in HBSS to further remove debris and hepatocytes. The isolated immune cells from the liver went through red blood cell lysis with ACK buffer (KD Medical) before counting cells on a hematocytometer. Splenocytes were isolated by passing cells through a 70 µm cell strained followed by red blood cell lysis with ACK buffer before being transferred to a 96-well U-bottom plate and resuspended in fluorescence-activated cell sorting (FACS) buffer (2% FBS in 1X PBS). Viability staining was performed using LIVE/DEAD fixable red stain (1 in 1000 in FACS buffer, Invitrogen) for 15 min at room temperature. Suspensions were then pelleted and resuspended in anti-CD16/32 antibodies (1:500, BioLegend) to block non-specific binding of Fc receptors. Cells were incubated with the indicated surface antibodies for 30 min at 4 °C. a FoxP3 transcription factor staining kit (eBioscience) was used for intracellular staining. Antibodies against intracellular proteins were diluted in 1X permeabilization buffer and added for 45 min at 4 °C. For cytokine staining, cells were stimulated with PMA (final concentration of 1 µg/mL) and ionomycin (Iono, Cell Signaling; final concentration of 1 µg/mL) for 4 h at 37 °C in the presence of brefeldin A (GolgiPlug, BD Biosciences; final concentration of 1 µg/mL) to block cytokine export from the golgi apparatus. 2% paraformaldehyde (PFA) was used to fix the cells after staining. Cells were resuspended in 100 µL 1X PBS and run on the FACSymphony A3 5-laser flow cytometer (BD Biosciences). Data were analyzed using FlowJo (v.10, BD Biosciences).

Formalin-fixed liver tissue was paraffin embedded, and sectioned using standard approaches. H&E, Picrosirius Red, and oil red-O staining was done by the SBP histology core using standard approaches and analyzed using Python 3.6.10. H&E images were scored by a trained pathologist (C.M.) for age-associated liver pathology. For Picrosirius Red, images were processed by applying a median filter to each RGB channel using a disk of size 2, then converted to HSV to isolate red hues with predefined thresholds. Noise was reduced by removing small objects, and the Picrosirius Red-stained areas were quantified as a ratio of stained area or intensity to the total non-white area. For oil red-O, images were processed using the OpenCV library for red droplet isolation via HSV color segmentation, followed by a 2 × 2 pixel morphological opening to refine droplet boundaries. Droplet count, size, and total area were quantified using skimage.measure.

No statistical method was used to predetermine sample size. Sample sizes for cell culture experiments were determined empirically on a per-experiment basis. Sample sizes for animal experiments were determined by initial pilot experiments. For data generated by automated imaging, images that were out of focus or that contained technical artifacts were removed. For processed data, obvious outliers were verified by Grubb's test and removed. For cohort 1 of the animal experiments, one young male mouse was excluded from data analysis due to the presence of an open wound and suspected infection at time of collection. The experiments were not randomized, except for animal experiments, in which animals were randomly assigned into treatment groups. The investigators were not blinded to sample allocation during cell culture experiments and outcome assessment. However, to limit bias, immunofluorescence imaging was done using automated image capture in NIS Elements AR v5.21.03 in a predetermined pattern for each sample, and all images were scored by NIS Elements AR v5.21.03 software to avoid bias of manual scoring, with the exceptions of Figs. 2F and 3D. Blinding was used for initial sample processing and analysis for animal experiments, except for Fig. 4A.Statistical significance for routine assays was calculated in Graphpad Prism 10 or Microsoft Excel (Fig. S1B, Fig. S2I), using p < 0.05 as a threshold. For cell culture experiments, pairwise comparisons were done by two-sided Student's t-test assuming unequal variance between groups. Simple linear regressions were calculated in Graphpad Prism 10. Ingenuity pathway analysis used a right-tailed Fisher's exact test to determine a p-value cutoff for overlap between gene lists. For animal experiments, pairwise comparisons were done using a Mann-Whitney U test, or by one-way ANOVA (Fig. 4H) in Graphpad Prism 10. Biological sex was considered in the animal study design, with data for both sexes shown in Fig. S5.