C57BL/6J mice were purchased from the Jackson Laboratory. All mice were housed in temperature-controlled rooms with a 12 h light-dark cycle and given free access to water and food. After starvation for 5 h, 8 weeks-old male mice were injected intraperitoneally with PBS, β-HB, or S-β-HB (1.5 g/kg body weight in PBS). In parallel, 8 weeks-old male mice were randomized and fed a regular chow diet or fasted for 72 h. Then, the mice were sacrificed. Tissues were collected for gene expression evaluation in various organs including the aorta, liver, heart, brain, adipose, and kidney (n = 6). All animal protocols were reviewed and approved by the Institutional Animal Care and Use Committee at Georgia State University.

Human umbilical vein endothelial cells (HUVECs, catalog no. LSC0035C) and human aortic smooth muscle cells (hASMCs, catalog no. C0075C), isolated from an individual donor were originally obtained from the Thermo Fisher Scientific (Waltham, MA, USA). HUVECs were cultured in EBM Basal Medium (Lonza, catalog no. CC-3121) supplemented with EGM SingleQuats (Lonza, catalog no. CC-4133) and hASMCs were cultured in Medium 231 (GIBCO, catalog no. M231500) supplemented with SMGS (GIBCO, catalog no. S00725). All culture media were supplemented with 5% heat-inactivated fetal bovine serum (FBS, GIBCO), 100 U mL -1 penicillin, and 0.1 mg mL -1 streptomycin (Sigma-Aldrich) in a 37°C incubator with humidity and 5% CO 2 . HUVECs and hASMCs were obtained from single donor. And mice primary cells were isolated from 6 mice/group at the same day. The cells were maintained and regularly tested for mycoplasma contamination by PCR and confirmed to be free of any contamination.

Method Details

SA β-galactosidase assay SA β-gal staining kit (Cell Signaling Technology, catalog no. 9860) was used to stain senescent cells. According to the manufacturer’s instructions (Cell Signaling Technology), HUVECs and hASMCs were fixed for 10 min, stained for 1 day and analyzed. Senescent cells, identified as blue-stained cells, were captured with light microscopy (Olympus IX73), then images were processed using imaging software (CellSens Dimension). Mice aortas were fixed for 10 min, stained for 1week and captured with light microscopy.

Fluorescein Di-β-D-Galactopyranoside assay (FDG) Fluorescein Di-β-D-Galactopyranoside (FDG) was used to quantify the SA β-gal activity in HUVECs. FDG was dissolved in DMSO solution in which the sock solution (FDG, 200 mM) was stored at −20°C. Cells were cultured in 96-well plates, washed with PBS, then fixed with solution (2% formaldehyde, 0.2% glutaraldehyde). Staining solution (37 mM citric acid, 126 mM Na 2 HPO 4 , 5 mM potassium ferricyanide, 5 mM potassium ferrocyanide, 150 mM NaCl, 2 mM MgCl 2 ) was added to each well, then 10 μL of 2 mM FDG was added to each well. After incubation in the dark at 37°C for 24 h without CO 2 supply, the supernatant was transferred to a 96-well plate and fluorescein fluorescence was measured using the fluorometer (Tecan M1000) with a 485 nm excitation and a 535 nm emission wavelength. Genomic DNA of attached cells was measured with Qubit dsDNA HS assay kit in order to normalize β-galactosidase activity.

SPiDer β-galactosidase assay Cells were washed with PBS, and SPiDer β-Gal working solution was added to the culture dish for 30 min at 37°C. After reaction, cells were washed with PBS, then fixed with 4% PFA/PBS solution. SA β-galactosidase positive cells were captured under fluorescence microscope with a 488 nm excitation and 533 nm emission wavelength. The mice aortas were separated into aortic arch and thoracic aorta to performed SPiDer β-gal assay. Aortic arch and thoracic aorta were embedded in optimum cutting temperature compound (OCT) compound. Then, 2.5 mm of aortic arch was sectioned every 0.5 mm from innominate artery to left subclavian, and 5 mm of thoracic aorta was sectioned every 1 mm from the middle portion of the descending thoracic aorta to diaphragm. Tissue slides were washed with PBS, and reacted by SPiDer β-gal solution for one hour at 37°C. After washing slides with PBS, SA β-galactosidase positive areas were captured under fluorescence microscope with a 488 nm excitation and 533 nm emission wavelength.

Synthesis of β-HB, Butyrate-conjugated beads, and S-β-HB salt (S)-(+)-4-Amino-3-hydroxybutyric acid was dissolved in coupling buffer (0.2 M NaHCO 3 , 0.5 M NaCl, pH 8.3), and prewashed NHS-activated Sepharose beads (10 mL) were added to the solution. The mixture was incubated in a 4°C shaker for 24 h. Then, the remaining NHS-activated groups were blocked with blocking buffer (0.5 M ethanolamine, 0.5 M NaCl, pH 8.3). β-HB and Butyrate-conjugated beads were washed with washing buffer (0.1 M Tris-HCl, pH 8.9). In order to prepare S-β-HB salt, (S)-hydroxybutyric acid was dissolved in acetone, then 0.1 mM of NaOH solution was added dropwise. Crystalized white power was washed by acetone and dried. 1H NMR (D 2 O): δ 4.07 (m, 1H), 2.33 (t, 1H), 2.21 (t, 1H), 1.12 (d, 3H).

Affinity chromatography Cultured HUVECs were washed with PBS and then homogenized with a 26-gauge syringe in binding buffer (10 mM Tris-HCl, pH 7.4, 50 mM KCl, 5 mM MgCl 2 , 1 mM EDTA, and 0.1 mM Na 3 VO 4 ). The cell lysate was centrifuged at 13,000 rpm for 20 min at 4°C, and the supernatant was collected. In total, 4 mg of protein was obtained. The cell lysate was precleared by incubation with non-conjugated Sepharose beads and then loaded into a 15 mL volume of β-HB or Butyrate-conjugated Sepharose beads. After washing with 3 volumes of washing buffer (0.1 M Tris-HCl, pH 8.9), affinity chromatography was performed with β-HB gradient elution buffer (0.1 M Tris-HCl, β-HB 0.5 mM–8 mM). Samples were concentrated and separated by SDS-PAGE and visualized by Coomassie Brilliant Blue staining. The samples were then digested with trypsin, and peptide tandem mass spectrometry analysis of the digested peptides was performed using a Matrix-assisted laser desorption/ionization (MALDI) time-of-flight mass spectrometer.

Real-time RT-PCR Tabled 1 Primer Forward (5′ to 3′) Reverse (5′ to 3′) Oct4A CGTGAAGCTGGAGAAGGAGAAGCTG CAAGGGCCGCAGCTTACACATGTTC Oct4B ATGCATGAGTCAGTG AACAG CCACATCGGCCTGTGTATAT IL-1α ATCAGTACCTCACGGCTGCT TGGGTATCTCAGGCATCTCC IL-6 CCAGCTATGAACTCCTTCTC GCTTGTTCCTCACATCTCTC IL-8 ATGACTTCCAAGCTGGCCGTGGCT TCTCAGCCCTCTTCAAAAACTTCTC SOX2 GGGAAATGGGAGGGGTGCAAAAGAGG TTGCGTGAGTGTGGATGGGATTGGTG KLF4 GTTTTGAGGAAGTGCTGAG CAGTCACAGTGGTAAGGTTT c-Myc GTTGGTCAGGCTGGTCTTGAA CATGCGCCTGTAATCCTAGCA Nanog ACCAGAACTGTGTTCTCTTCCACC GGTTGCTCCAGGTTGAATTGTTCC GAPDH CAACTTTGGCATTGTGGAAGG ACACATTGGGG GTAGGAACAC

Immunofluorescence staining After β-HB treatment to the HUVECs, hASMCs or prepared thoracic aorta tissue of mice, samples were fixed with 4% paraformaldehyde (v/v) in PBS for 12 min at room temperature (RT), permeabilized with 0.2% Triton X-100 (v/v) in PBS for 10 min at RT, and blocked with 5% normal goat serum for 30 min at RT. Cells were incubated with primary antibodies (1: 500) at 37°C for 30 min, then followed by Alexa Fluor® 488 or 647 conjugated secondary antibodies (1: 500) at 37°C for 60 min. Images were captured using confocal microscope or fluorescence microscope.

Western blotting Cell lysates were prepared in RIPA lysis buffer (50 mM Tris, pH 7.0, 150 mM NaCl, 5 mM EDTA, 1% deoxycholic acid, 0.1% SDS, 30 mM Na 2 HPO 4 , 50 mM NaF, and 1 mM NaVO 4 ) containing protease inhibitor cocktail (Roche Applied Science). Proteins (40 μg) were resolved by 8 or 12% SDS-PAGE and transferred to PVDF membranes (Millipore). The membrane was blocked with 5% nonfat dry milk in TBS-T (50 mM Tris-HCl, pH 7.6, 150 mM NaCl, and 0.1% Tween-20) and probed with primary antibodies overnight at 4°C. The secondary antibodies used were horseradish peroxidase-conjugated goat anti-rabbit or anti-mouse IgG (Cell Signaling Technology). The membranes were washed 3 times with TBS-T and then visualized with chemiluminescent peroxidase reagents (Roche, Germany).

Subcellular fractionation Cells were harvested and homogenized by pass through a 27 gauge needle 10-times with fractionation buffer (150 mM KCl, 25 mM Tris pH 7.4, 5 mM EDTA, 0.5 mM, protease inhibitors). Centrifuge the cell lysate at 800 × G for 10 min. The pellet contains nuclei proteins and the supernatant contains cytoplasmic, mitochondrial and plasma-membrane proteins. Centrifuge supernatant at 10,000 × G for 20min. The pellet contains mitochondrial proteins and the supernatant contains cytoplasmic and plasma-membrane proteins. Finally, Centrifuge supernatant at 100,000 × G for 1 h. The supernatant contains pure cytoplasmic proteins and the pellet contains plasma-membrane proteins.

RNA-immunoprecipitation (RIP) Cytoplasmic proteins were obtained through subcellular fractionation method described in Method part. Cell lysate was resuspended in freshly prepared RIP buffer (150 mM KCl, 25 mM Tris pH 7.4, 5 mM EDTA, 0.5 mM DTT, 0.5% NP40, 100 U/mL RNAase inhibitor, and protease inhibitor). Antibodies to hnRNP A1 were added to supernatant and incubated for overnight at 4°C with gentle rotation. Protein A/G beads were added, then incubated for 2 h at 4°C with gentle rotation. Precipitated beads were washed three times and resuspended with RIP buffer. Bound RNAs were extracted with TRIzol according to manufacturer’s instruction. Synthesis of cDNA was conducted using iScript cDNA Synthesis Kit according to the manufacturer’s instruction. Gene expression was quantified on the CFX96 real-time system (Bio-Rad) using the iQ SYBR Green supermix (Bio-Rad).

Cell cycle analysis HUVECs and hASMCs were trypsinized from the culture dish after β-HB treatment and gathered by centrifugation. Precipitated cells were washed twice by repeating suspension and precipitation in PBS buffer. Precipitated cells were suspended with 500 μL PBS and fixed with 5 mL of ice-cold 70% ethanol overnight. Fixed cells were harvested and resuspended in PBS and treated with 100 μg/mL of RNase A at 37°C for 20 min. Propidium iodide (PI) was then added at a final concentration of 50 μg/mL for DNA staining, and 10,000 fixed cells were analyzed on a FACS Fortessa (BD Biosciences). For G0 phase analysis, Pyronin Y and Hoechst 33342 were used to stain mRNA and DNA separately. Cells were harvested in the same manner as described above, then re-suspend in culture media containing 10 μg/mL Hoechst 33342, incubated at 37°C for 15 min, then 5 μL of 100 ug/mL Pyronin Y was added. After a 15 min incubation, cells were transferred to ice and 10,000 cells were analyzed on a FACS Fortessa (BD Biosciences).

Oct4 knockdown by CRISPR-Cas9 Oct4A knockout HUVECs were prepared by CRISPR-Cas9-mediated gene editing. We used CRISPR design tool ( http://tools.genome-engineering.org ) to select Oct4 target sequences. Potential target oligomers for sgRNA constructs was designed with high efficiency score. Oct4 sgRNA sequence was 5′-CACCGGCACTAGCCCCACTCCAACC-3′ and 5′-AAACGGTTGGAGTGGGGCTAGTGCC-3′. Oct4 targeting gRNA was inserted into PX459 plasmid, then HUVECs were transfected with this plasmid. After transfection, the cells were selected with medium containing 2.0 μg/mL Puromycin.