Ab1101

BMSCs were analysed by western blotting based on our previous study [13 (link)]. For the preparation of total cell lysates, BMSCs were lysed in RIPA buffer (P0013B; Beyotime, Shanghai, China) at 4 °C. The samples were centrifuged, and the protein concentrations were checked using the Enhanced BCA Protein Assay kit (P0010S, Beyotime). The supernatants were separated on a 12% SDS-PAGE gel and subsequently transferred to a PVDF membrane (Immobilon-P Membrane, Millipore, USA). The membranes were blocked in 5% bovine serum albumin (BSA) for 1 h and then incubated with the appropriate primary antibodies overnight at 4 °C. Then, the membranes were incubated with the appropriate horseradish peroxidase-conjugated secondary antibody for 1 h at 24 °C. Immunoreactive bands were revealed by the BeyoECL Plus reagent (P0018, Beyotime) using the Photo-Image System (Molecular Dynamics, Sunnyvale, CA, USA). β-Actin was used as a loading control for the western blotting analysis. The following antibodies were used to analyse protein expression levels by western blotting: HJURP (ab224076, Abcam), GDF15 (ab39999, Abcam), CDKN1A (ab109520, Abcam) and p53 (ab1101, Abcam).

Proteins were extracted from the VAT and SCAT of macaques and ASC monolayers, as described previously [9 (link)], and then electroblotted on a nitrocellulose membrane (Biorad Laboratories, Richmond, CA, USA). Specific proteins were detected by incubation with specific primary antibodies for p16 (BD Pharminogen, Franklin Lakes, NJ, USA, 51-1325GR, dilution 1/500), phosphorylated-p53 (Abcam, Cambridge, UK, ab3897, dilution 1/1200), total p53 (Abcam, ab1101 dilution 1/1200), phospho-Akt (Ser473, Cell Signaling, Danvers, MA, USA, cs9271, dilution 1/1000), Akt (Cell signaling, cs9272, dilution 1/1000) and tubulin (Sigma, T5168, dilution 1/10,000), and then with horseradish-peroxidase-conjugated secondary antibodies at 1/5000 of dilution except for tubulin, the secondary antibody was diluted at 1/10,000. Immune complexes were detected by enhanced chemiluminescence (Amersham, GE Healthcare Europe, Velizy-Villacoublay, France).

Cells were lysed with a buffer containing 1% Triton X-100, 50 mM HEPES (pH 7.5), 150 mM NaC1, 10% glycerol, 1.5 mM MgCl₂, 5 mM EGTA, protease inhibitors (4 mM phenyl methylsulfonyl fluoride and 100 mg/ml aprotinin, Sigma-Aldrich), and phosphatase inhibitors (10 mM sodium orthovanadate and 20 mM sodium pyrophosphate, Sigma-Aldrich) and processed. For direct immunoblot analysis, we employed 15–30 μg of total cellular proteins, which were resuspended with 25 μl of loading buffer, boiled for 5 min, and loaded on SDS-PAGE for Western blot (WB). The antibodies for WB were used at the condition suggested by the suppliers: rabbit anti-NOTCH-1 (ab27526, 1/500, Abcam, UK), rabbit anti-Bcl2 (Ab185002, 1/500, Abcam), rabbit anti-human NUMB (ab-14140, 1/1000, Abcam), mouse anti-p53 (ab1101, 1/1000, Abcam), mouse anti-Myc (sc-40, 1/200, Santa Cruz Biotechnology, Texas, USA), and mouse anti-beta-actin (sc-81178, 1/1000, Santa Cruz Biotechnology). The WBs were acquired with the ChemiDoc MP Imaging System (Bio-Rad Laboratories Inc., California, USA), and the corresponding bands were quantified with Image Lab 6.1.0 (Bio-Rad Laboratories, Inc.). The p-value for the relative amount was obtained with the Student’s t-test.

To analyze the first mitotic divisions in vitro, keratinocytes were isolated from freshly obtained adult and aged human skin samples using Dispase 25 U/ml, followed by 0.05% trypsin‐EDTA, and keratinocytes (unselected/unsorted) were plated in chamber slides with 154CF medium (0.07 mM calcium chloride) (Thermo Fisher Scientific). Twenty‐four hours later, cells were fixed with 3% paraformaldehyde, incubated with anti‐p53 (DO‐1, ab1101), anti‐Notch1 (ab4498b), anti‐Numb (ab4147), and anti‐keratin 1 (ab93652) primary antibodies (Abcam), followed by either AlexaFluor 488 or 568 secondary antibodies (Invitrogen). Divisions were identified as closely associated post‐mitotic sister pairs (forty to 100 divisions were analyzed for each sample). Greater than 98% of closely associated post‐mitotic sister pairs were BrdU+ at the employed seeding density, confirming cell division (Charruyer et al., 2016, 2017).

Nuclear foci secondary to DNA Double-Strand Breaks (DSBs) were visualized by immunofluorescence with antibodies directed against Ser139-phosphorylated histone variant H2A (γ-H2AX, 05-636, Merk, Sigma-Aldrich) in transduced HCAECs. Nuclear DNA was stained with di-amidino-2-phenylindole hydrochloride staining (DAPI, Sigma-Aldrich).
The blue staining produced by SA-β-galactosidase’s hydrolysis of 5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside (X-Gal, Sigma-Aldrich) was used as a biomarker of cellular senescence [21 (link)]. To quantify SA-β-galactosidase activity, cells were incubated with appropriate buffer solution containing X-Gal at pH 6, as described previously [22 (link)]. The proportion of positive (blue) cells was determined. The protein expression of cell cycle arrest markers was detected by incubation with specific primary antibodies for p53 (ab1101, Abcam, Cambridge, UK) and p21 (#2947, Cell Signaling, Danvers, MA, USA), as previously described [20 (link)].

The cells were lysed using protease inhibitor-contained radioimmunoprecipitation assay buffer (R0010, Solarbio, P.R. China) for protein extraction. After sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis, the protein was subsequently transferred onto polyvinylidene fluoride membranes and probed with the following primary antibodies (Abcam, Cambridge, UK): mouse anti-KDM3A (1:1,000, ab91252), mouse anti-phosphorylated (p-)53 (1:1,000, ab1101), rabbit anti-cleaved caspase-3 (1:500, ab49822), rabbit anti-p53-k372me (1:10,000, ab16033), rabbit anti-caspase-3 (1:2,000, ab13847), and mouse anti-β-actin (1:10,000, ab8226). Western blots were exposed to horseradish peroxidase-coupled goat anti-rabbit IgG (ab205718, 1:20,000) or goat anti-mouse IgG (ab205719, 1:20,000) and enhanced chemiluminescence detection reagents (BB-3501, Amersham Pharmacia Biotech, Little Chalfont, UK). Target protein bands were quantified using Quantity One v4.6.2 software, with β-actin used for normalization.