Chemical compounds and cytokines

Commercially available compounds and various cytokines used in the study are listed in Supplementary Table 2. The synthesis and analysis of PZ15227 (PZ) and Bcl-xl-NP are presented in Supplementary Note 1.

Cell culture

Human WI38 fibroblasts (WI38, Cat. No. CCL-75), human IMR90 fibroblasts (IMR90, Cat. No. CCL-186), human renal epithelial cells (RECs, Cat. No. PCS-400-012) and human pre-adipocytes (PACs, Cat. No. PCS-210-010) were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA). WI38 and IMR90 cells were cultured in complete Dulbecco’s modified Eagle medium (DMEM, Cat. No. 12430054, Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% heat-inactivated fetal bovine serum (FBS, Cat. No. S11150H, Atlanta Biologicals, Flowery Branch, GA, USA), 100 U/mL penicillin and 100 µg/mL streptomycin (Pen Strep, Cat. No. 15140122, Thermo Fisher Scientific) in a humidified incubator at 37 °C and 5% CO 2 . RECs were cultured in renal epithelial cell basal medium (Cat. No. PCS-400-030, ATCC) supplemented with complements from renal epithelial cell growth kit (Cat. No. PCS­400­040, ATCC). PACs were cultured in fibroblast basal medium (Cat. No. PCS-201-030, ATCC) supplemented with fibroblast growth kit-low serum (Cat. No. PCS­201­041, ATCC).

SC induction

SCs were induced and validated as previously described13,14,16. Briefly, low-passage WI38 cells (<25 passages), IMR90 cells (<25 passages), RECs (<10 passages), and PACs (<4 passages) were used as NCs or for the induction of senescence. To induce SCs by ionizing radiation (IR-SCs), WI38, and IMR90 fibroblasts, RECs and PACs were treated as previously described12,13,14,16. To induce replicative senescence (RE-SCs), WI38 cells were subcultured until they stopped to divide and became permanently growth arrested or senescent after about 37 passages. To induce cellular senescence by ectopic expression of H-Ras (Ras-SCs), WI38 cells were infected with lentivirus containing pLenti CMV/TO RasV12 Puro (w119-1) (catalog no. 22262, Addgene, Cambridge, MA, USA). Two days after viral infection, transfected cells were selected by 2 μg/mL puromycin. Five days later, cells became senescent.

Western blotting

After treatment with PZ or indicated compounds, cells were harvested in 1.5 mL microcentrifuge tubes and washed twice with ice-cold PBS. Protein was extracted using the RIPA buffer with EDTA and EGTA (Cat. No. BP-115DG, Boston BioProducts, Ashland, MA, USA) supplemented with 1% protease inhibitor cocktail (Cat. No. P8340, Sigma-Aldrich, St. Louis, MO, USA). Samples were put on ice for 30 min and then kept at −80 °C freezer overnight. After centrifugation at 15,000 × g at 4 °C for 20 min, supernatant was collected and the protein concentration was determined using the Pierce BCA protein Assay kit (Cat. No. 23225, Thermo Fisher Scientific). An equal amount of proteins (20–40 µg/lane) was loaded to a precast gel (Mini-PROTEAN TGX, Cat. No. 456-1094, Bio-Rad, Hercules, CA, USA) and transferred onto PVDF membranes (Invitrolon, Cat. No. LC2002, Life Technologies, Carlsbad, CA, USA) by electrophoresis. The membranes were blocked with 1X TBS-Tween (TBST, Cat. No. J77500, Affymetrix, Santan Clara, CA, USA) containing 5% non-fat dry milk (Cat. No. sc-2324, Santa Cruz Biotechnology, Dallas, TX, USA), and subsequently probed with primary antibodies at a predetermined optimal concentration overnight at 4 °C. After washing with TBST for 3 times (10 min each time), the membranes were incubated with the secondary horse radish peroxidase (HRP)-linked antibody for 1–2 h at room temperature. Following sufficient washing with TBST, the membranes were incubated with chemiluminescent HRP substrate (Cat. No. WBKLS0500, MilliporeSigma, Billerica, MA, USA). The blotting membranes were recorded using autoradiography (SRX-101, Konica, Shinjuku, Tokyo, Japan) or the ChemiDoc MP Imaging System (Bio-Rad, Hercules, CA, USA). Information on all antibodies used in western blot analyses is listed in Supplementary Table 3. The immunoblots were quantified using the ImageJ version 1.52a software.

Cell viability assay

Number of viable cells was quantified using flow cytometry as previously described13,14,16. Specifically, NCs and SCs were incubated with vehicle (0.1% DMSO) or a test compound at indicated concentrations for indicated time points. Following dissociation with 0.25% Trypsin-EDTA (Cat. No. 25200056, Thermo Fisher Scientific) at 37 °C for 3–5 min, cells were harvested in 100 µL pre-cooled phosphate-buffered saline (PBS, Cat. No. 20012027, Thermo Fisher Scientific) containing 2% FBS and 100 ng/mL propidium iodide (PI, Cat. No. P4170, Sigma-Aldrich) and analyzed using flow cytometry (LSR II, BD Biosciences, San Jose, CA, USA). BD FACSDiva Software was used to collect the data. Percentage of viable cells were determined by counting the number of PI negative cells (viable cells) and then calculated as a ratio of control cells treated with vehicle13,14,16. Dose-response curves were generated for each compound, and the concentration for 50% of maximal effect (EC 50 values) was calculated using GraphPad Prism 7 (GraphPad Software, La Jolla, CA, USA).

Platelet isolation and viability assay

Human platelet-rich plasma (PRP) was purchased from Zenbio (Cat. No. SER-PRP-SDS, Research Triangle Park, NC, USA) within 3 days after harvest. PRP was transferred into a 50 mL tube containing 5 mL acid citrate buffer (Cat. No. sc-214744, Santa Cruz Biotechnology, Dallas, TX, USA). Mouse platelets were isolated from 10 to 16-week-old C57BL/6 J mice. Whole mouse blood was collected into a 50 mL polypropylene (PP) tube containing 5 mL citrate-dextrose solution (ACD) solution (Cat. No. sc-214744, Santa Cruz Biotechnology, Dallas, TX, USA) after euthanization. The anticoagulated blood was centrifuged at 250 × g for 20 min at room temperature without break. PRP was collected into a 15 mL polypropylene conical tube. To prevent clotting, prostaglandin E1 (PGE1, Cat. No. sc-201223A, Santa Cruz Biotechnology) and apyrase (Cat. No. A6237, Sigma-Aldrich) were added to final concentrations of 1 µM and 0.2 units/mL, respectively. After gently mixing the solution, platelets were pelleted by centrifugation at 1200 × g for 10 min without break. Pelleted platelets were gently washed without disrupting platelets in 2 mL HEPES Tyrode’s buffer (Cat. No. PY-921WB, Boston BioProducts, Ashland, MA, USA) containing 1 µM PGE1 and 0.2 units/mL apyrase. After washing, pellets were slowly suspended in 10 mL HEPES Tyrode’s buffer containing 1 µM PGE1, 0.2 units/mL apyrase and 10% FBS. Platelets number was then counted using the HEMAVET 950FS hematology analyzer (Drew Scientific, Miami Lakes, FL, USA). For viability assays, platelet number was adjusted to 2 × 108/mL in HEPES Tyrode’s buffer containing 1 µM PGE1, 0.2 units/mL apyrase and 10% FBS. Each treatment was given in 2 mL platelet suspension in 15 mL polypropylene tubes. The tubes were placed on a rotating platform at room temperature and the viability of platelets was measured after treatment for indicated time points by using the MTS reagent (Cat. No. G1111, Promega, Madison, WI, USA). Through the whole process of the platelet viability assay, extra cautions such as room temperature storage/incubation, centrifuge without break, gently pipetting and mixing, and rotating platelets during incubation were taken to avoid platelet activation, aggregation, and spontaneous apoptosis. The data were analyzed by GraphPad Prism 7 software for the calculation of EC 50 values.

AlphaLISA assay

In order to evaluate the binding affinities of PZ and ABT263 towards Bcl-2, Bcl-xl, and Bcl-w, the AlphaScreen competitive binding assay was performed at room temperature and all reagents were diluted in a buffer containing 25 mM HEPES pH 7.5, 100 mM NaCl, 0.1% BSA, and 0.005% Tween-20. Purified recombinant His-tagged Bcl-xl, His-tagged Bcl-2 and His-tagged Bcl-w (Cat. No. SRP0187 for Bcl-xl, Cat. No. SRP0186 for Bcl-2 and Cat. No. B1059 for Bcl-w, Sigma-Aldrich) were incubated with increasing concentrations of compounds and a fixed concentration of biotin-tagged BAD (Cat No. SQ-ASPE-66592-1, AnaSpec, Fremont, CA) (for Bcl-xl) or BIM (Cat. No. SQ-ASPE-66592-2, AnaSpec) (for Bcl-2 and Bcl-w) peptides to a final volume of 40 µL in 96-well PCR plates. After 24 h incubation, 5 µL 6X His-Acceptor beads (final concentration 20 µg/mL) (Cat. No. AL128M, Perkin-Elmer, Waltham, MA, USA) were added to each well and incubated for 1 h. Thereafter, 5 µL streptavidin-donor beads were added (final concentration of 20 µg/mL) (Cat. No. 6760002, Perkin-Elmer) to each well and incubated for 30 min. At the end of the incubation period, 17 µL of each sample was transferred into adjacent wells of 384-well proxy plate (Cat. No. 6008280, Perkin-Elmer). The plate was scanned using Alpha program on a Biotek’s Synergy Neo2 multi-mode plate reader (Winooski, VT, USA). Gen5 version 3.04 software (BioTek, USA) was used for the AlphaLISA signal measurements. The inhibition constant (Ki) was calculated using non-linear regression, one site, competitive binding, fit Ki function on GraphPad Prism 7 software based on experimentally determined Kd for each protein/peptide pair.

Apoptosis analysis

WI38 NCs and SCs were treated with vehicle or 10 µM Q-VD-OPh (QVD, Cat. No. S7311, Selleckchem, Houston, TX, USA) for 4 h prior to the addition of vehicle or PZ for 72 h. The suspended cells were first harvested from the culture and then pooled with the adherent cells detached by 0.25% Trypsin-EDTA at 37 °C for 3–5 min in 12 × 75 mm polystyrene round-bottom tubes (Cat. No. 352058, Falcon, Corning, NY, USA) containing complete DMEM medium. The cells were stained with Alexa Fluor 647-Annexin V (1: 50, Cat. No. 640912, BioLegend, San Diego, CA, USA) and PI (10 µg/mL, Cat. No. 421301, BioLegend, San Diego, CA, USA) at room temperature for 30 min. For analysis of platelet apoptosis, human platelets were washed with PBS containing 2% FBS and stained with Alexa Fluor 647 Annexin V (1:20) for 20 min at room temperature. The stained cells were analyzed on an Aurora flow cytometer and SpectroFlo Software was used to collect the data (Cytek Aurora, Fremont, CA, USA).

CRBN knockout by CRISPR/Cas9 genomic editing

To knockout CRBN, the sgRNA targeting human CRBN was designed and cloned into a lentiCRISPR v2 vector (a gift from Feng Zhang; Addgene plasmid # 52961). Packaging 293 T cells were transfected with CRBN sgRNA or negative controls (non-targeting sgRNA-NC)47 and helper vectors (pMD2.G and psPAX2; Addgene plasmid #s 12259 and 12260) using Lipofectamine 2000 reagent (Cat. No. 11668019, Life Technologies). Medium containing lentiviral particles and 8 µg/mL polybrene (Cat. No. H9268, Sigma-Aldrich,) was used to infect WI38 cells. Infected cells were selected in medium containing 2 μg/mL puromycin. The target guide sequences are as follows: sgCRBN: forward (5′-CACCGAACCACCTGCCGCTCCTGCC-3′) and reverse (5′-AAACGGCAGGAGCGGCAGGTGGTTC-3′).

Quantitative PCR (qPCR)

Total RNA was extracted using RNeasy Mini kit (Cat. No. 74106, QIAGEN, Gaithersburg, MD, USA). A total of 500–1000 ng mRNA was reversely transcribed into cDNA using the High Capacity cDNA Reverse Transcription kits (Cat. No. 4368813, Life Technologies) according to the manufacturer’s instructions. cDNA was then diluted with RNase free water by 5 times. Human GAPDH, mouse Hprt or mouse Mrps2 was used as internal controls. Four μL diluted cDNA was mixed with 10 µL TaqMan Fast Advanced Master Mix (Cat. No. 4444965, Thermo Fisher Scientific) and 1 µL of Taqman assay probe (Supplementary Table 4). Samples were added 5 µL of H 2 O to have 20 µL of mixture and run qPCR according to the manufacturer’s instructions. All reactions were run in duplicate on an ABI StepOnePlus Real-Time PCR System (Applied Biosystems, Foster City, CA, USA). Target gene expression was calculated by normalizing to the housekeeping genes using the ∆Ct method48.

Ubiquitination assay

Plasmid pSG5-Flag-Bcl-xl (pDL2009) was constructed by using Gibson Assembly Master Mix (NEB, Ipswich, MA, USA) and the following two primers sets:

primer set-1, forward (5′-GACTACAAGGACGACGATGACAAGGGATCTATGTCTCAGAGCAACCGGGAGCTGGTG-3′) and reverse (5′-GTTCTGCTTTAATAAGATCTGGATCTTCATTTCCGACTGAAGAGTGAGCCCAGCAGAAC-3′);

primer set-2, forward (5′-GTTCTGCTGGGCTCACTCTTCAGTCGGAAATGAAGATCCAGATCTTATTAAAGCAGAAC-3′) and reverse (5′-CACCAGCTCCCGGTTGCTCTGAGACATAGATCCCTTGTCATCGTCGTCCTTGTAGTC-3′).

pLX304-Bcl-xl (DNASU plasmid # HsCD00437924) and pSG5 (obtained from Invitrogen and Stratagene) were used as templates. Flag-Bcl-2 plasmid was a gift from Clark Distelhorst (Addgene plasmid #18003, Watertown, MA, USA). HA-Ub plasmid was a gift from Ted Dawson (Addgene, Plasmid #17608, Watertown, MA, USA). HEK293T cells (1.6 × 106) were co-transfected with Flag-Bcl-xl and HA-Ub or Flag-Bcl-2 and HA-Ub for 40 h and then treated with DMSO, 1 μM ABT263 or 1 μM PZ15227 for 4 h. MG132 (10 μM) was added to prevent protein degradation in all cultures. Proteins were extracted by using immunoprecipitation lysis buffer (Cat. No. 87788, Thermo Fisher Scientific) and then subjected to immunoprecipitation using Anti-FLAG M2 Magnetic Beads (Cat. No. M8823, Sigma-Aldrich) according to the manufacturer’s protocol. Anti-FLAG M2 Magnetic Beads was washed with 1X TBS three times and then added to protein samples, and the mixture was incubated at 4 °C with rotation overnight. The magnetic beads were collected and then washed three times with 1X TBS. Immunoprecipitated samples were eluted with 2X SDS sample buffer and boiled 5 min at 95 °C. Proteins were analyzed by western blotting as described above.

Naturally aged mice and treatments

Young (about 2 months old) male C57BL/6 J (or CD45.2) mice and B6.SJL-PtprcaPep3b/BoyJ (or CD45.1) mice (used as recipients for hematopoietic stem cell transplantation) were purchased from Jackson Lab (Bar Harbor, MA, USA). Breeding pairs of p16-3MR transgenic mice were kindly provided by Dr. Judith Campisi (Buck Institute for Research on Aging, Novato CA)49 and bred and maintained in the AAALAC-certified animal facility at the University of Arkansas for Medical Sciences (UAMS) and University of Florida (UF). They and their progeny received food and water ad libitum. Mice with tumors and/or leukemia were excluded from experiments and analyses. For the experiments presented in Figs. 3–6, 20-month-old male and female mice were randomly assigned to one of the treatment groups and received IP injections of vehicle (0.1 mL/mouse, q3d, 7 injections, n = 8 mice), ABT263 (41 μmole/kg or 40 mg/kg/q3d, 7 injections, n = 6 mice), or PZ (41 μmole/kg or 61 mg/kg/ q3d, 7 injections, n = 7 mice). ABT263 and PZ were formulated in 50% PHOSAL 50 PG, 45% MIGLYOL® 810 N and 5% Polysorbate 80. A group of untreated young (2 months old, n = 8 mice) p16-3MR transgenic mice was included as controls. Various tissues were harvested for analyses after the mice were euthanized by CO 2 suffocation and cervical dislocation 6 days after receiving the last injection. For the experiments presented in Supplementary Fig. 8, both male and female mice were randomly assigned to one of the treatment groups and received IP injections of vehicle (0.1 mL/mouse, 7 days per cycle for 2 cycles with a 2-week interval, n = 5 mice), ABT263 (41 μmole/kg or 40 mg/kg/day × 7 days per cycle for 2 cycles with a 2-week interval, n = 5 mice), or PZ (41 μmole/kg or 61 mg/kg/day × 7 days per cycle for 2 cycles with a 2-week interval, n = 5 mice). A group of untreated young (2 months old, n = 5 mice) p16-3MR transgenic mice was included as controls. Tissues were harvested for analyses after the mice were euthanized by CO 2 suffocation and cervical dislocation 2 days after receiving the last injection.

TBI mice and treatments

p16-3MR transgenic mice at 2–3 months of age were exposed to sham irradiation as controls or a sublethal dose (6.5 Gy) of TBI in a X-RAD 320 irradiator (Precision X-Ray, Branford, CT, USA) at a dose rate of 82 cGy/min. Fifteen weeks after TBI, four mice were treated with vehicle (50% PHOSAL 50 PG, 45% MIGLYOL® 810 N and 5% Polysorbate 80), four mice with ABT263 (41 μmole/kg or 40 mg/kg, q3d/i.p), and five mice with PZ15227 (41 μmole/kg or 61 mg/kg, q3d/i.p.) for 3 weeks. A group of untreated (2–3 months old, n = 5 mice) p16-3MR transgenic mice was included as controls (CTL). Various tissues were harvested for analyses from above animal models after the mice were euthanized by CO 2 suffocation and cervical dislocation 6 days after receiving the last injection.

In vivo platelet toxicity assay

Female 5–6-week-old C57BL/6 mice were purchased from Jackson Lab (Bar Harbor, MA, USA) and treated with single IP injection of ABT263 (40 mg/kg) or PZ15227 (40 and 61 mg/kg). Approximately 50 μL of blood was collected from each mouse at 6 h, day 1, day 3, day 5, day 7, and day 10 via submandibular plexus route in EDTA tubes (Cat. No. 077051, RAM Scientific, Inc. Nashville, TN, USA) and platelets were enumerated using an automated hematology analyzer HEMAVET 950FS (Drew Scientific, Miami Lakes, FL, USA). For the mice used in Figs. 3–6 and Supplementary Fig. 7, mice were treated with indicated concentrations of ABT263 or PZ15227. Twenty-four hours after the first and last treatments, ~50 μL of blood was collected from each mouse into EDTA tubes through via submandibular plexus, and complete blood counts (CBCs) including platelets were immediately enumerated using HEMAVET 950FS (Drew Scientific, Miami Lakes, FL, USA).

All mice were housed in the Assessment and Accreditation of Laboratory Animal Care (AAALAC)-accredited animal facilities at UAMS or UF under pathogen-free conditions. All animal work was approved and done in accordance with the approvals from the Institutional Animal Care and Use Committees of UAMS and UF with the exception of the pharmacokinetic (PK) studies that were done by BioDuro (San Diego, CA, USA), a global contract research organization, through a contract. All animal studies were complied with the ethical regulations and humane endpoint criteria according to the NIH Guidelines for the Care and Use of Laboratory Animals.

Analysis of blood T, B, and myeloid cells by flow cytometry

Blood T cells (CD3e+), B cells (B220+), and myeloid (M) cells (CD11b/Gr-1+) were stained with anti-CD3e, B220, Gr-1 and CD11b antibodies and analyzed on an Aurora flow cytometer (Cytek Aurora)16,50. Dead cells were excluded by gating out the cells stained positive with 7-Aminoactinomycin D (7-AAD, Cat. No. A1310, Thermo Fisher Scientific). The information for all these antibodies used in the staining was provided in Supplementary Table 5.

Phenotypic analysis of BM cells by flow cytometry

The femora and tibiae were harvested from the mice immediately after they were euthanized. Bone marrow (BM) cells were flushed from the bones into PBS containing 2% FBS using a 21-gauge needle and syringe. BM mononuclear cells (BM-MNCs) were isolated and stained with the appropriate antibodies presented in Supplementary Table 5 to measure the frequencies of different cell types in BM, i.e., lineage negative (Lin−) cells, hematopoietic progenitor cells (HPC or Lin−sca1−c-kit+cells), LSK cells (Lin−sca1+c-kit+cells), and Scal1−c-Kitlow cells, HSC (Lin−sca1+c-kit+CD48–CD150+ cells), granulocyte–monocyte progenitors (GMP or Lin–sca1–c-kit+CD34+CD16/32+), megakaryocyte–erythrocyte progenitors (MEP or Lin–sca1–c-kit+CD34–CD16/32–), common myeloid progenitors (CMP or Lin–sca1–c-kit+CD34+CD16/32–) and common lymphoid progenitors (CLP or Lin–Sca1–c-Kitlow CD127+CD135+) using an Aurora flow cytometer (Cytek Aurora)16,50. FlowJo version 10 software was used to analyze the flow cytometry data.

Isolation of BM HSCs

For the isolation of Lin–cells, BM-MNCs were incubated with biotin-conjugated rat antibodies specific for murine CD3e, CD11b, CD45R/B220, Ter-119, and Gr-1. The labeled mature lymphoid and myeloid cells were depleted by incubation with goat anti-rat IgG paramagnetic beads (Dynal Inc, Lake Success, NY, USA) at a bead: cell ratio of ~4:1. Cells binding the paramagnetic beads were removed with a magnetic field. The negatively isolated Lin– cells were washed with 2% FBS/PBS, resuspended in 2% FBS/PBS at 1 × 107/mL. LSK, HSCs and long-term HSCs were stained and isolated according the methods described previouly16,50. Dead cells were excluded by gating out 7-AAD (1:100, final concentration 10 μg/mL) positive cells. The sorted HSCs and LT-HSCs were cultured for single-cell colony-forming assay and competitive repopulation assay, respectively, as described below. The information for all these antibodies used in the staining was provided in Supplementary Table 5.

Colony-forming cell assay in single HSC liquid culture

Single HSCs from individual mice were directly sorted into wells of round-bottom 96-well plates. The cells were cultured in freshly prepared RPMI 1640 culture medium supplemented with 10% FBS, 1% BSA, 1% penicillin and streptomycin, 2 mM L-Gln, 5 × 10–5 M β-mercaptoethanol, 10 ng/mL of recombinant mouse stem cell factor (mSCF), thrombopoietin (mTPO), and interleukin-3 (mIL-3). After 14 days of culture, the numbers of cells produced by each HSC were counted and the cells that produced more than 10,000 cells were scored as colony-forming cells. The information for mSCF, mTPO, and mIL-3 was provided in Supplementary Table 2.

Competitive repopulation assay (CRA)

Fifty freshly sorted LT-HSCs from mice treated with vehicle or PZ were mixed with 3 × 105 competitive BM cells pooled from three young CD45.1 mice and then transplanted into lethally irradiated (9.5 Gy TBI) CD45.1 recipients via retro-orbital injection of the venous sinus. Donor cell engraftment in the recipients was analyzed at 4 months after transplantation16.

Senescence-associated-β-galactosidase (SA-β-gal) staining

BM LSK (Lin−sca1+c-kit+) cells were isolated according to the above protocol. SA-β-gal staining in LSK cells was performed by flow cytometry using an ImaGene Green C12FDG lacZ gene expression kit from Molecular Probes (Cat. No. I2904, Thermo Fisher Scientific) according to the manufacturer’s instructions and modified as previously described16,50,51.

Osteoblast and adipocyte differentiation assays

Total BM cells were obtained by flushing the tibiae and femurs and pooled from more than 2–3 mice from each group. To analyze osteoblast progenitor cell activity, BM stromal cells were cultured with 20% FBS, 1% penicillin and streptomycin and 50 µg/mL of ascorbic acid (Cat. No. A4403, Sigma-Aldrich) in 10-cm culture dishes for 7 days. Half of the medium was replaced every 3 days. BM stromal cells were then re-plated in 12-well plates with 10% FBS, 1% penicillin and streptomycin, 50 µg/mL of ascorbic acid and 10 mM glycerophosphate (Cat. No. G9422, Sigma-Aldrich) for 5 days to perform qPCR assays or 21 days to perform Alizarin Red S staining. Mineralized matrix was stained with 40 mM alizarin red solution, following the manufacturer’s instructions (Cat. No. 40-1009, Sigma-Aldrich).

To analyze adipogenic potential, BM stromal cells were cultured to 80% confluence, and the media supplemented with rosiglitazone (5 nM/mL) or with 3.3% BSA in PBS as vehicle control. Medium was changed every 3–4 days. After 10 days, cells were fixed in 10% formalin in PBS, rinsed, and stained for 30 min with 0.15% Oil Red O (Cat. No. O0625, Sigma-Aldrich) in a 55:45 mix of isopropanol and water. Cells were counterstained with 0.5% methyl green (Cat. No. M295-25, Fisher Scientific, Pittsburgh, PA, USA) in 0.1 M sodium acetate, pH 4. Oil Red O staining was quantified after extraction of the dye with 1 mL isopropanol and absorbance determination at 490 nm. For all assays, cells were plated in triplicate.

Osteoclast differentiation assay

BM cells were harvested as described above. After the red blood cells were removed with ACK buffer (0.01 mM EDTA, 0.011 M KHCO 3 and 0.155 M NH 4 Cl, pH 7.3), we suspended the cells in α-MEM complete media containing 10% FBS, 1% penicillin and streptomycin and incubated the cells for 24 h in the presence of 10 ng/mL of M-CSF. Non-adherent BM myeloid were re-plated in Petri dishes with 30 ng/mL of M-CSF for 4 days to generate BM macrophages (BMMs), which were used as osteoclast precursors. To generate mature osteoclasts, BMMs were cultured with 30 ng/mL of M-CSF and 30 ng/mL of RANKL for 4–5 days. The medium was replaced every 3 days. Osteoclasts were fixed with 10% neutral buffered formalin for 15 min and stained for tartrate-resistant acid phosphatase (TRAP), using the Leukocyte Acid Phosphatase Assay Kit (Cat. No. 386 A, Sigma-Aldrich), following the manufacturer’s instructions. An osteoclast was defined as a multinuclear (more than 3 nuclei) TRAP-positive cell. For all assays, cells were plated in triplicate.

SA-β-gal staining in BM stromal cells

SA-β-gal activity was detected using the Senescence β-Galactosidase Staining Kit (Cat. No. 9860, Cell Signaling Technology, Danvers, MA, USA) according to the manufacturer’s instructions.

Statistical analysis

All statistical analyses were performed with Graphpad Prism 7. All data are presented as means ± SEM (or SD if the data are from replications of a representative experiment but not from independent assays). Comparisons were made by two-tailed Student’s t-test when comparing two experimental groups. For a comparison between more than two groups, one-way ANOVA with Tukey’s or Dunnett’s post-hoc tests was used. For one-way ANOVA analysis that failed the normality test, Kruskal–Wallis one-way ANOVA with Dunn’s post-hoc test was performed. For Fig. 2h–j, two-way ANOVA with Tukey’s post-hoc tests was used. All western blots showed in the figures were repeated in at least two independent experiments. P < 0.05 was considered to be significant. The exact P values were provided in the Source Data file.

Reporting summary

Further information on research design is available in the Nature Research Reporting Summary linked to this article.