Synthesis of SSK1

Unless otherwise mentioned, all reactions were carried out under a nitrogen atmosphere with dry solvents under anhydrous conditions. Reagents were purchased at the highest commercial quality and used without further purification, unless otherwise stated. Yields were measured chromatographically.

Reactions were monitored by thin-layer chromatography on plates (GF254) supplied by Yantai Chemicals (China) using UV light as the visualizing agent and an ethanolic solution of phosphomolybdic acid and cerium sulfate and heat as the developing agents. Unless otherwise specified, flash column chromatography used silica gel (200–300 mesh) supplied by Tsingtao Haiyang Chemicals (China).

NMR spectra were recorded on a Brüker Advance 600 (13C 150 MHz, 19F 565 MHz) and a Brüker Advance 400 (1H 400 MHz) spectrometers, which were calibrated using residual undeuterated solvent (CD 3 OD at 3.31 ppm 1H NMR, 49.0 ppm 13C NMR). The following abbreviations were used to explain the multiplicities: s = singlet, d = doublet, t = triplet, dd = doublet of doublets, m = multiplet.

Mass spectrometric data were obtained using a Brüker Apex IV FTMS using ESI (electrospray ionization).

Gemcitabine (2.0 equiv, 0.340 mmol, 89.5 mg) and DIPEA (2.4 equiv, 0.402 mmol, 70 μL) were dissolved in 4 mL of DMF. The mixture was stirred for 20 min in a microwave reaction vessel before addition of 1 (1 equiv, 0.170 mmol, 113 mg).62 The reaction was placed in a CEM Discover microwave reactor (100 °C) and irradiated for 45 min. Then, DMF was removed in vacuo, and the residue was purified by flash column chromatography to give the desired product SSK1 (39.5 mg, 29% yield) as a white solid.

R f = 0.3 (dichloromethane: acetone = 2:1); 1H NMR (400 MHz, CD 3 OD): δ 8.31 (d, J = 7.6 Hz, 1 H), 7.90 (s, 1 H), 7.69 (d, J = 8.6 Hz, 1 H), 7.47 (d, J = 8.6 Hz, 1 H), 7.31 (d, J = 7.6 Hz, 1 H), 6.24 (t, J = 7.2 Hz, 1 H), 5.47 (d, J = 3.4 Hz, 1 H), 5.39 (d, J = 3.3 Hz, 1 H), 5.38 (s, 1 H), 5.27–5.24 (m, 3 H), 4.35 (t, J = 6.5 Hz, 1 H), 4.32–4.26 (m, 1 H), 4.21 (d, J = 6.4 Hz, 2 H), 3.99–3.94 (m, 2 H), 3.81 (dd, J = 12.9, 3.2 Hz, 1 H), 2.18 (s, 3 H), 2.08 (s, 3 H), 2.04 (s, 3 H), 1.97 (s, 3 H); 13C NMR (150 MHz, CD 3 OD) δ 172.01, 171.93, 171.41, 171.26, 165.22, 157.29, 154.18, 150.26, 145.72, 142.22, 134.66, 132.87, 125.72, 123.89 (t, J = 258.9 Hz), 119.81, 101.03, 97.02, 86.34 (t, J = 32.0 Hz), 82.76 (t, J = 3.8 Hz), 72.58, 71.96, 70.14 (t, J = 23.0 Hz), 69.50, 68.56, 66.90, 62.60, 60.25, 20.69, 20.65, 20.50, 20.49; 19F NMR (565 MHz, CD 3 OD) δ -119.06 (dd, J = 239.7, 9.8 Hz, 1 F), −119.91 (d, J = 241.7 Hz, 1 F); HRMS-ESI (m/z): calc’d for C 31 H 35 F 2 N 4 O 18 [M + H+] 789.1909, found 789.1907.

Cell culture

The mouse primary cells (MEFs, NBFs, and adult mouse lung fibroblasts) were cultured in high-glucose DMEM (Gibco) supplemented with 10% fetal bovine serum (FBS, VISTECH) and 1% penicillin-streptomycin (Gibco). HEFs were cultured in medium Dulbecco’s modified Eagle’s medium (DMEM, Gibco) containing 10% fetal bovine serum (FBS, VISTECH), 1% GlutaMAX (Gibco), 1% Non-Essential Amino Acids (NEAA, Gibco) and 1% penicillin/streptomycin (Gibco). HUVECs were cultured in Endothelial Cell Medium (ECM, sciencecell) with 10% FBS and 1% penicillin/streptomycin (Gibco). Human preadipocytes were cultured in Mesenchymal Stem Cell Growth Medium 2 (PromoCell, C-28009). All the cells were all cultured in a humidified incubator at 37 °C and 5% CO 2 .

Mouse embryonic fibroblasts (MEFs) were isolated from E13.5 embryos as described previously.63 Newborn mouse skin fibroblasts (NBFs) were isolated from the skin of day 1–3 newborn mice. Adult mouse lung fibroblasts were isolated from 3-month-old mice. Briefly, mouse embryonic tissues, skin and lung tissues were obtained from described donor mice. Then, these tissues were minced with forceps and incubated in 2 mg/mL collagenase IV (Gibco) for 2–4 h at 37 °C. After enzyme treatment, cells were collected by centrifugation and resuspended in high-glucose DMEM (Gibco) supplemented with 10% (VISTECH) and 1% penicillin-streptomycin (Gibco).

For human primary fibroblast isolation, the present study was approved by the Research Ethics Committee of China-Japan Friendship Hospital (Ethical approval No: 2009-50) and the Institute of Review Board in Peking University (IRB 00001052-1508719070) and conducted according to the approved protocol. Samples were collected from consenting donors according to ethically approved procedures at China–Japanese Friendship Hospital. Human embryonic skin fibroblasts (HEFs) were obtained as previously described.64 Briefly, human embryonic skin tissues, obtained from aborted tissues with informed patient consent, were minced with forceps and incubated in 1 mg/mL collagenase IV for 1–2 h at 37 °C. After enzyme treatment, cells were collected by centrifugation and resuspended in HEF medium (DMEM containing 10% FBS, 1% GlutaMAX, 1% NEAA and 1% penicillin/streptomycin). Cells were plated on 10 cm tissue culture dishes and grown in HEF medium.

HUVECs were purchased from Lonza (C2519A) and cultured in Endothelial Cell Medium (sciencecell, 1001). HUVECs were subcultured and cryopreserved according to the protocol from Lonza.

Human preadipocytes, were obtained as previously described.65 Briefly, human adipose tissues were collected from patients during liver or gallbladder surgery. The samples were washed with phosphate-buffered saline (PBS) containing 5% Penicillin/Streptomycin (P/S) to remove visible blood vessels and debris. Then, the tissues were minced with forceps and digested with 1 mg/mL Collagenase Type IV containing 2% P/S for 2–4 h at 37 °C, 5% CO 2 . After enzyme treatment, cells were collected by centrifugation and resuspended in DMEM (Gibco) containing 10% fetal bovine serum (FBS, VISTECH) and 1% penicillin/streptomycin (Gibco). Cells were plated on 10 cm tissue culture dishes and grown in Mesenchymal Stem Cell Growth Medium 2 (PromoCell, C-28009).

Cellular senescence induction

To avoid replication-induced senescent cells, low-passage proliferative NBFs (<3 passages), MEFs (<3 passages), mouse lung fibroblasts (<3 passages), HEFs (<10 passages), HUVECs (<6 passages), and human preadipocytes (<5 passages) were used as normal controls.

For replication-induced senescence, cells were passaged until they lost the ability to proliferate and became senescent. NBFs and MEFs were used as senescent cells after approximately 8–9 passages or 12 population doublings. Mouse lung cells were senescent after approximately 7 passages or 10 population doublings. HEFs were used after approximately 35 passages or 50 population doublings. HUVECs were senescent after approximately 18 passages or 24 population doublings. Human preadipocytes were senescent after approximately 10 passages or 15 population doublings.

For ionizing radiation-induced senescence, MEFs and HEFs were exposed to 10 Gy of ionizing irradiation in an RS 2000 X-ray Biological Irradiator (Rad Source Technologies, Inc.) at a dose rate of 1.205 Gy/min. The day after irradiation, MEFs, HEFs and HUVECs were passaged at a 1:2 dilution and cultured for another three days. Then these cells were plated to carry out further experiments.

For oncogene-induced senescence by ectopic expression of KRAS, cells were transfected with lentivirus carrying pLenti-PGK-KRAS4B (G12V) or control vector (Addgene, 35633). One day after viral infection, the medium was exchanged, and transfected cells were cultured for another 3 days and passaged at a 1:3 dilution. After another 3 days of culture, the cells became senescent enough for further analysis.

For etoposide-induced senescence, MEFs were treated with 2 µM etoposide for 12–18 h, and HEFs were treated with 5 µM etoposide for 12–18 h. Three days after treatment, cells were sub-cultured at a 1:3 ratio for 3 days in a fresh medium before further analysis.

For peroxide hydrogen (H 2 O 2 )-induced senescence, cells were incubated in medium with 200 μM H 2 O 2 for 4 h or 100 μM for 12 h and cultured in fresh medium for several days until they lost the capacity of proliferation.

Cell viability testing

To test the viability of senescent and non-senescent cells treated with SSK1, cells were stained with a mixture of Hoechst 33342 and propidium iodide (PI) (Solarbio, CA1120) to distinguish living and dead cells. After 3 days of treatment with the small molecules, the cell culture medium was removed, and the cells were washed once with PBS. Then, the cells were stained according to the manufacturer’s protocol, where the final concentrations of Hoechst 33342 and PI were 5 μg/mL and 2 μg/mL in PBS buffer. The plate was incubated at 4 °C for 30 min and observed on a fluorescence microscope or automatic cell imaging system. Viable cells were quantified using a MD Image Xpress Micro XL (Molecular Devices). The treatments include the following small molecules: gemcitabine (MedChem Express, HY-17026), SSK1 alone, SSK1 with SB203580 (MedChem Express, HY-10256), SSK1 with SB202190 (MedChem Express, HY-10295), SSK1 with Birb796 (MedChem Express, HY-10320), SSK1 with Dipyridamole (Yuanye, B25482), LY2334737 (Targetmol, T4061), 4-(Hydroxymethyl)-2-nitrophenol (Bidepharm, BD97717), ABT-263 (Selleck, S1001), Dasatinib (Selleck, S1021) plus Quercetin (Selleck, S2391), Fisetin (Selleck, S2298).

FDA-approved drug screening

Replication-induced senescent Newborn mouse skin fibroblasts (NBFs) were seeded in 24-well plates and treated with an FDA-approved drug screening library (selleckchem). Compounds in the library were diluted to a final concentration of 1 μM. After 3 days of treatment, the cell viability was tested.

SSK1 metabolism analysis

To detect the release of gemcitabine from SSK1 in senescent and non-senescent cells, the metabolism analysis was performed as previously reported.66 Briefly, the senescent and non-senescent cells were incubated with 0.5 μM SSK1 in DMEM medium containing 10% FBS and 1% penicillin-streptomycin for the indicated time. Then the cells were slightly washed with PBS for three times, then harvested and counted. Cold methanol used to extract the compounds and samples were prepared after centrifugation. The concentration of gemcitabine was determined by LC-MS/MS analysis.

Flow cytometry analysis

To test apoptosis of cells treated with Vehicle or SSK1 by flow cytometry analysis, NBFs were digested into single-cell suspension by incubation with 0.25% trypsin-EDTA (Invitrogen) at 37 °C for 3–5 min or with accutase at 37 °C for 10 min. Cells were then stained with FITC annexin V and propidium iodide (PI) according to the manufacturer’s protocol of Annexin V-FITC Apoptosis Detection Kit (Beyotime Biotechnology, C1063). Flow cytometry was performed within 1 h on an Aria III (BD Biosciences) or FACSVerse (BD Biosciences). Data were analyzed using FlowJo software (FlowJo LLC). Viability was calculated as the percentage of PI and Annexin V double-negative cells. Apoptotic cells were Annexin V-positive cells.

To test the SA-β-gal activity by flow cytometry analysis, senescent and non-senescent cells were digested into single cells by trypsinization, and then incubated with 33 μM 5-dodecanoylaminofluorescein di-β-D-galactopyranoside (C12FDG) (Invitrogen, I-2904) for 20–60 min. Pre-treatment with chloroquine diphosphate may inhibit endogenous β-galactosidase activity. The cells were washed with PBS after staining and the cell suspensions were run immediately in a FACSVerse (BD Biosciences). Data were analyzed using FlowJo software (FlowJo LLC).

To test β-gal-positive cell types in bleomycin-induced lung injury model and mice kidneys and salivary glands by flow cytometry analysis, we first treated the isolated primary cells with 33 μM C12FDG-containing culture medium for 20–60 min. Then these cells were co-stained by cell type markers on ice for 45 min. The monoclonal conjugated antibodies were used: anti-CD45 PerCP-Cy5.5 (1:200, eBioscience, 45-0451-82), anti-CD326 (EpCAM) PE-Cy7 (1:200, eBioscienc, 25-5791-80). DAPI (Beyotime, C1002) was used for live/dead discrimination. All samples were acquired and sorted using an FACSVerse (BD Biosciences). Data were analyzed using FlowJo software (FlowJo LLC).

Cell proliferation assays

For BrdU staining, cells were labeled with BrdU (5 μM in culture medium) (Sigma-Aldrich, B5002) for 3–12 h and then fixed with 4% paraformaldehyde (DingGuo, AR-0211). Cells were incubated in 2 M HCl for 1 h and neutralized with 0.1 M sodium borate buffer pH 8.5 for 15 min. BrdU incorporation was measured by immunofluorescence staining (IF).

For EdU staining, cells were labeled with EdU (10 μM) for 3 h and stained according to the manufacturer’s protocol (Beyotime, C0071S). BrdU or EdU incorporation was quantified by high content analysis microscopy — MD Image Xpress Micro XL (Molecular Devices).

Reverse transcription (RT)-quantitative PCR (RT-qPCR)

Total RNA was isolated using the Direct-zol RNA MiniPrep Kit (Zymo Research, R2072). RNA was treated with DNase and converted to cDNA using TransScript First-Strand cDNA Synthesis SuperMix (TransGen Biotech, AT311–03). RT-qPCR was performed using Kapa SYBR® FAST qPCR Kit Master Mix (Kapa Biosystems, KM4101) on a CFX ConnectTM Real-Time System or CFX96TM Real-Time System (Bio-Rad). Data were analyzed using the 2(−ΔΔCt) method. GAPDH was used as a control to normalize the expression of target genes.

Primers for specific genes of mouse

Gene Forward Primer (5′ to 3′) Reverse Primer (5′ to 3′) p16 GCCCAACGCCCCGAACTCTTTC GCGACGTTCCCAGCGGTACACA p21 CCTGGTGATGTCCGACCTG CCATGAGCGCATCGCAATC p19 GCCGCACCGGAATCCT TTGAGCAGAAGAGCTGCTACGT IL1α AAGTCTCCAGGGCAGAGAGG CTGATTCAGAGAGAGATGGTCAA IL1β AAAAGCCTCGTGCTGTCG AGGCCACAGGTATTTTGTCG IL6 GTTCTCTGGGAAATCGTGGA GGTACTCCAGAAGACCAGAGGA CXCL1 ACCGAAGTCATAGCCACACTC CTCCGTTACTTGGGGACACC TNFα GCCTCTTCTCATTCCTGCTT CTCCTCCACTTGGTGGTTTG GLB1 GGATGGACAGCCATTCCGAT CAGGGCACGTACATCTGGATA Fn1 CCACCCCCATAAGGCATAGG GTAGGGGTCAAAGCACGAGTCATC Col1a1 TGCCGTGACCTCAAGATGTG CACAAGCGTGCTGTAGGTGA Col3a1 GCGGAATTCCTGGACCAAAAGGTGATGCTG GCGGGATCCGAGGACCACGTTCCCCATTATG GAPDH CTTTGTCAAGCTCATTTCCTGG TCTTGCTCAGTGTCCTTGC

Primers for specific genes of human

Gene Forward Primer (5′ to 3′) Reverse Primer (5′ to 3′) p16 ATATGCCTTCCCCCACTACC CCCCTGAGCTTCCCTAGTTC GAPDH TGACATCAAGAAGGTGGTGAAGCAGG GCGTCAAAGGTGGAGGAGTGGGT

Plasmid construction and lentivirus production

Plasmids used to interfere the expression of GLB1 with short hairpin RNAs (shRNAs) were obtained from Sigma MISSION shRNA. Genetic knockdown was performed according to the manufacturer’s protocol. The shRNA sequences are listed below. Lentivirus production, collection, and infection were as described.67

shRNA sequences

shRNA Sequences (5′ to 3′) shGLB1#1 CCGGGAGAAGTCATTCAGATGTTTACTCGAGTAAACATCTGAATGACTTCTCTTTTTTG shGLB1#2 CCGGGTGCTAGAATGGAAGCTACATCTCGAGATGTAGCTTCCATTCTAGCACTTTTTTG shGLB1#3 CCGGGAGAAGTCATTCAGATGTTTACTCGAGTAAACATCTGAATGACTTCTCTTTTTTG shControl CCGGCAACAAGATGAAGAGCACCAACTCGAGTTGGTGCTCTTCATCTTGTTGTTTTTG

Western blot

Senescent and non-senescent NBFs were plated and incubated overnight at 37 °C. The cells were treated with 0.05 μM gemcitabine or 0.5 μM SSK1 for the indicated time. Before harvest, cells were washed twice with pre-cooled PBS buffer. Total protein was extracted with lysis buffer (50 mM Tris-HCl (pH 7.5), 137 mM sodium chloride, 1 mM EDTA, 1% Nonidet P-40, 10% glycerol, 0.1 mM sodium orthovanadate, 10 mM sodium pyrophosphate, 20 mM β-glycerophosphate, 50 mM sodium fluoride, 1 mM phenylmethylsulfonyl fluoride), and the protein concentrations were normalized using the BCA Protein Assay Kit (Thermo Fisher Scientific, 23225). Protein samples were mixed with protein loading buffer and incubated at 95 °C for 5 min. Western blot was performed by using the following antibodies: phospho-specific p38 MAPK (Thr180/Tyr182) (Cell Signaling Technology, 4511) and p38 (Cell Signaling Technology, 8690); phospho-MKK3(S189)/MKK6(S207) (Cell Signaling Technology, 9231); cleaved caspase 3 (Cell Signaling Technology, 9661). The β-tubulin or β-actin protein level was also determined as the loading control by using the β-tubulin antibody (Cell Signaling Technology, 2128) or β-actin antibody (Cell Signaling Technology, 4970).

Mitochondrial DNA analysis

About 0.5 × 107 senescent or non-senescent cells were treated with 0.5 μM SSK1 or DMSO in this assay. After treatment, the cells were washed with PBS and collected for mitochondrial DNA extraction by using the kit (Abcam, ab65321). The concentration of mitochondrial DNA was measured by nanodrop. Then 500 ng mitochondrial DNA from each sample was used and separated by 1% agarose gel for analysis.

Mice

All animal experiments were performed according to the Animal Protection Guidelines of Peking University, China. C57BL/6 (C57) mice were obtained from Beijing Vital River Laboratory Animal Technology Co, Ltd. and maintained under specific pathogen-free facility (SPF) conditions with a 12 light/12 dark cycle and free access to food and water. Young mice were 2–4-month-old; middle-aged mice were 9–12-month-old; old mice were 20–24-month-old. Old male mice were caged individually, and female mice and young male littermates were maintained with no more than five mice per cage.

Bleomycin-induced lung injury

Bleomycin was purchased from Selleck (S1214). To induce lung senescence and fibrosis, male and female mice (3–6-month-old) were subjected to transtracheal injection of bleomycin (1.5 mg/kg) as previously reported.68

Drug treatments

All drugs were mixed in 90% PBS, 5% Tween-80 (Sigma, P1754), and 5% polyethylene glycol (PEG) (Sigma, 81172) and administered to mice by intraperitoneal (i.p.) injection. For bleomycin-treated young mice, SSK1 (0.5 mg/kg) or vehicle were given consecutive two days every week for four weeks. For old mice (20-month old), SSK1 (0.5 mg/kg), gemcitabine (0.5 mg/kg), ABT263 (5 mg/kg), dasatinib (1 mg/kg) plus quercetin (10 mg/kg), fisetin (20 mg/kg) or vehicle (DMSO) were administrated for continued 3 days every 2 weeks for 8 weeks. Mice were randomized to SSK1, gemcitabine or vehicle delivered by intraperitoneal injection.

Toxicity testing in vivo

Old mice were injected with vehicle and SSK1 (3, 10, 30, 60, 100 mg/kg) 3 times a week for 5 weeks. After treatment, the blood samples were collected for further analysis.

SA-β-galactosidase staining of cultured cells and frozen sections

Cultured cells were washed once with PBS and fixed in SA-β-galactosidase (SA-β-gal) staining fix solution for 15 min at room temperature. Cells were then washed three times with PBS and incubated with SA-β-gal staining solution (Beyotime Biotechnology, C0602) for 16–20 h at 37 °C. For 12-well plates, 1 mL of β-gal staining solution was added to each well. The plate was sealed with Parafilm to prevent evaporation of the staining medium. After the overnight incubation, cells were washed with PBS and observed under a bright field microscope.

For SA-β-gal staining, the frozen sections were dried at 37 °C for 20–30 min and then fixed in SA-β-gal staining fix solution for 15 min at room temperature. The frozen sections were washed three times with PBS and incubated with SA-β-gal staining solution (Beyotime Biotechnology, C0602) overnight at 37 °C. After completion of SA-β-gal staining, the sections were stained with eosin for 1–2 min, rinsed under running water for 1 min, differentiated in 1% acid alcohol for 10–20 s, and washed again under running water for 1 min. Sections were dehydrated in increasing concentrations of alcohols and cleared in xylene. Excess xylene was drained and a coverslip was placed over the section. After drying, the sample was observed under a microscope.

Lung, liver, and kidney frozen sections stained with SA-β-gal were quantified by ImageJ software (NIH) to measure the SA-β-gal-positive area. The total area was quantified by eosin-positive area. The relative SA-β-gal-positive cells were calculated with the SA-β-gal-positive area divided by the total area. For the statistics of SA-β-gal-positive area of lung, the regions of lung NBFs were randomly selected to be photographed, avoiding analysis of larger pulmonary blood vessels and trachea. For the statistics of SA-β-gal-positive area of liver, the regions were randomly selected to be photographed. For the statistics of SA-β-gal-positive area of kidney, the regions of renal cortex were randomly selected to be photographed. Each tissue was measured with 10–18 regions.

β-galactosidase of frozen sections

For endogenous acidic β-galactosidase (β-gal) staining, the kidney and salivary gland frozen sections were dried at 37 °C for 20–30 min and then fixed in β-gal staining fix solution for 15 min at room temperature. The frozen sections were washed three times with PBS and incubated with β-gal staining solution (Beyotime Biotechnology, C0605) overnight at 37 °C. The following protocol was similar to that of the SA-β-gal staining.

Masson’s trichrome stain

To test the fibrosis in lung or liver tissues, the Masson’s trichrome staining kit (Solarbio, G1340) was used. The formalin-fixed, paraffin-embedded sections of lungs or livers were defaraffinized with xylene and rehydrated through 100% alcohol, 95% alcohol and running tap water. Then the sections were stained according to the manufacturer’s instruction. The sections were dehydrated very quickly through 95% alcohol and 100% alcohol, cleared by xylene and then covered with cover slides. The images were collected using a bright-light microscope (10X magnification objective) (OLYMPUS, BX43) and analyzed by ImageJ software (NIH) to measure the fibrosis area and total area. Each tissue was measured with 5 different regions. The percentage of fibrosis was calculated with the fibrosis-positive area divided by the total area.

Immunofluorescence staining

Mouse tissues were fixed in 4% paraformaldehyde (DingGuo, AR-0211) for 24 h at room temperature and dehydrated with graded sucrose solution (20% and 30% respectively 24 h) before embedded in OCT compound (Sakura, 4583) for cryosection. The embedded tissues were cut and affixed on slides. Before immunohistochemical staining, the sections were fixed in 4% paraformaldehyde (DingGuo, AR-0211) at room temperature for 15 min and blocked with PBS containing 0.3% Triton X-100 (Sigma-Aldrich, T8787) and 2% normal donkey serum (Jackson ImmunoResearch Laboratories, 017-000-121) at room temperature for 1 h. Samples for immunofluorescence staining were incubated with primary antibody at 4 °C overnight, washed three times with PBS and then incubated with appropriate secondary antibodies for 1.5 h at 37 °C. Nuclei were stained with Hoechst 33342 (Sigma-Aldrich, B2261).

To identify the cell type of SA-beta-gal-positive cells, we co-stained SA-beta-gal with different cell type markers by immunofluorescence staining, the primary antibodies used in this study including: anti-F4/80 antibody (Abcam, ab6640) for detecting macrophage, anti-S100A4 (Fibroblast-specific protein 1, FSP1) antibody (Abcam, ab197896) for detecting fibroblasts, anti-EpCAM antibody (Abcam, ab71916) for detecting epithelial cells, anti-CD31 antibody (Abcam, ab28364) for detecting endothelial cells. To identify senescent cells, the primary antibody p21 (Abcam, ab188224) was used. To identify proliferative cells, the primary antibody Ki67 (MilliporeSigma, AB9260) was used.

The secondary antibodies were Alexa Fluor® 488 AffiniPure Donkey Anti-Rabbit IgG (H + L) (Jackson ImmunoResearch Laboratories, 71-545-152), Alexa Fluor® 488 AffiniPure Donkey Anti-Rat IgG (H + L) (Jackson ImmunoResearch Laboratories, 712-545-150), and Cy™3 AffiniPure Donkey Anti-Rabbit IgG (H + L) (Jackson ImmunoResearch Laboratories, 711-165-152). For quantification of p21-positive cells, images were randomly taken at 20× magnification at the same exposure using confocal laser endomicroscopy (LSM 710 NLO &DuoScan System) and then analyzed by ImageJ. Each mouse was measured with at least 10 regions.

RNA sequencing

Total RNA was isolated using Direct-zol RNA MiniPrep Kit (Zymo Research). RNA sequencing libraries were constructed using the NEBNext Ultra RNA Library Prep Kit for Illumina (NEB England BioLabs). Fragmented and randomly primed 2 × 150 bp paired-end libraries were sequenced using Illumina HiSeq X Ten. RNA sequencing and raw data quality control were performed by Novogene Co., Ltd.

The sequencing data quality were checked by FastQC (version 0.11.8, http://www.bioinformatics.babraham.ac.uk/projects/fastqc/). Sequencing data were aligned to the mm10 reference genome by TopHat2. For differential gene expression analysis, read count of each gene was obtained by HTSeq69 (version 0.11.1, https://htseq.readthedocs.io/en/release_0.11.1/). The leading log-fold-change of different conditions which was calculated by R package edgeR70 (version 0.38.0, https://bioconductor.org/packages/release/bioc/html/edgeR.html) was used as input for gene set enrichment analysis. The gene sets of mouse molecular signature were obtained by msigdbr (version 7.0.1, https://cran.r-project.org/web/packages/msigdbr/index.html). In this package, the original human genes of Molecular Signatures Database (MSigDB v7.0, http://software.broadinstitute.org/gsea/msigdb/index.jsp) were converted to non-human model organism homologous genes. Gene set enrichment analysis was performed by clusterProfiler71 (version 3.14.0) (https://bioconductor.org/packages/release/bioc/html/clusterProfiler.html). R (version 3.6.0, https://cran.r-project.org/) were used for gene expression analysis.

Gene ontology (GO) term enrichment analyses for Supplementary information, Fig. S7a-f were performed using DAVID 6.8 functional annotation tool.72 The gene lists were selected by comparing gene expression between old and young mice or SSK1-treated and vehicle-treated old mice with t-test statistics: fold changes > 2 and P values < 0.05. The top five Go terms of GO_Biological Progress and GO_Cellular Component were shown in Supplementary information, Fig. S7a–f.

ELISA analysis

Mouse blood samples were collected, stewed 2 h at room temperature or overnight at 4 °C, and then centrifuged (3000 rpm, 10 min) to gain serum. Secretion of mouse IL1α, IL6, CXCL1 and TNFα was measured using Mouse Interleukin 1α (IL1α) ELISA Kit (CUSABIO, CSB-E04621m), Mouse Tumor Necrosis Factor α (TNFα) ELISA Kit (CUSABIO, CSB-E04741m) kit, Mouse chemokine (C-X-C motif) ligand 1 (CXCL1) ELISA kit (CUSABIO, CSB-E17286m) and IL-6 (Interleukin-6) Mouse ELISA Kit (Abcam, ab100712) according to the manufacturers’ instructions.

Physical function measurements

All functional assays were conducted at least 5 days after the last dose of drug was administered.

The rotarod test was used to evaluate motor coordination and balance with an accelerating RotaRod system (SANS Bio Instrument, SA102). Mice were placed in separate lanes on the rod rotating at an initial speed of 4 rpm/min. The apparatus was set to accelerate from 4 to 44 rpm/min in 300 s. A timer was used to record when each mouse fell or clung to the rod and completed a full passive rotation. Mice were trained at least two times on days 1 and 2 and tested on days 3, 4, and 5. Results were the average over 3 trials.

Treadmill exhaustion tests were used to evaluate exercise capacity and endurance. A motorized treadmill was used at an incline of 5° with 0.5 mA electrical stimulation (SANS Bio Instrument, SA102). Mice were trained for three days, starting at an initial speed at 5 m/min for 2 min and accelerating to 7 m/min for 2 min and then 9 m/min for 1 min. After three training sessions and one day of rest, mice were tested on the fifth day at an initial speed of 5 m/min, which increased by 2 m/min every 2 min until mice were unable to return to the treadmill. The distance (m) traveled before exhaustion was recorded for each mouse.

Mice were placed on the top of a grid strength meter (Columbus Instruments, 1027DM), so they grasped the grid with all four paws. The meter was set to the Peak Tension (T-PK) mode and recorded the grip strength over seven trials. The grip strength (N) was averaged, with the maximum and minimum data points excluded.

Mice were placed on a 1-m long, 6-mm wide beam resting 60 cm above the floor. A black box full of nesting material from the home cage was placed at the end of the beam apparatus as the end point. At the first day, mice were trained three times to walk across the beam to the safe box successfully without hesitation or observation. On the test day, the time (s) to cross the center 80 cm mark was measured by two motion detectors: one at 0 cm that starts a timer and one at 80 cm that stops the timer.

Mice were taken from the housing room into the testing room and allowed to acclimate to the new environment for a minimum of 30 min before the test. Mice were carefully placed in a 14-cm high and 11-cm diameter transparent cylinder and recorded for 5 min by video camera. The resulting video was analyzed to measure the rearing frequency (when mice stand only on hind legs, raise forelimbs off the ground, and stand upright for over 1 s).

Blood analysis

For blood routine examination, 50 μL fresh blood was collected from each mouse and mixed with EDTA immediately. The blood samples were analyzed by Celltac Alpha MEK-6400 series hematology analyzers (Nihon Kohden, MEK-6400). For serum biochemical analysis, blood samples were collected, clotted for 2 h at room temperature or overnight at 4 °C, and then centrifuged (1000× g, 10 min) to obtain serum. 200 μL serum was aliquot and analyzed of Alanine transaminase (ALT), aspartate transaminase (AST), uric acid (UA) and creatinine (CREA) by Chemistry Analyzer (Mindray, BS-350E).

TUNEL Apoptosis analysis

To test the off-target effect of SSK1 on tissues, we examined the apoptosis of cells in kidneys. The experiment was performed according to the manufacturer’s protocol (Beyotime, C1090).

Statistical analyses

For statistical analyses, P values were calculated by t-test (when comparing only two groups) or one-way ANOVA or two-way ANOVA (when comparing more than two groups) in Excel or GraphPad Prism 8 with default parameters. All results are expressed as the mean ± SEM, and n indicates the number of experiment replicates or the number of mice. P values are as follows: *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.