Cyclin a

H₂O₂ (35 wt%) was purchased from Sigma−Aldrich (St. Louis, MO, USA). D−gal (≥99.0%) was obtained from Solarbio (Beijing, China). NMN (≥98.0%) and MET (≥98.0%) were obtained from Yuanye Biotechnology Co., Ltd. (Shanghai, China). According to a previously used method, SFE was isolated and purified [85 (link),86 (link)]. Primary antibodies against cleaved caspase−9, caspase−9, cleaved caspase−3, caspase−3, Cyclin D1, CDK6, Cyclin A, CDK2, Nrf2, HO−1, NQO−1, GCLM, TLR4, MyD88, p−NF−κB p65, p−IκB, and IL−6 were purchased from Cell Signaling Technology (Danvers, MA, USA). An antibody against β−actin was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Secondary antibody HRP−conjugated goat anti−rabbit IgG and goat anti−mouse IgG were obtained from Abcam (Cambridge, MA, USA). Maintenance feed for mice (#SPF−F02−001) was purchased from SPF (Beijing) Biotechnology Co., Ltd.

Res was purchased from LC Laboratories (Woburn, MA, USA). Sor was purchased from Selleck Chemicals (Houston, TX, USA). RPMI 1640 medium and fetal bovine serum (FBS) were purchased from Hyclone (GE Healthcare, USA). Cell Counting Kit-8 (CCK-8) was obtained from Dojin Chemical Co. (Kumamoto, Japan). Annexin V-FITC/propidium iodide (PI) apoptosis assay kit was bought from Roche (Roche, USA). PI and other reagents were purchased from Sigma-Aldrich (Sigma, USA). Antibodies to CDK2, CDC25A, CyclinA, caspase-3, caspase-8, caspase-9, PKA, eEF2K, p-AMPK, and AMPK were purchased from Cell Signaling Technology (Massachusetts, USA).

Recombinant antibodies against IRS-4, αp85 were obtained from Upstate Biotechnology (Lake Placid, NY, USA). Antibodies against ERK 1/2, p-AKT (Thr 308), p-Rb (ser 807/811), Rb, E2F1, cyclin A, cyclin B, cyclin D1, cyclin E, cdk 2, cdk 4, p-cdk1, FAK, p-FAK (Tyr 925, Tyr 397), and β-tubulin were from Cell signaling Technology Inc. (Danvers, MA). Antibodies against p-ERK, p-AKT (Ser473), p-Tyr (PY99), and integrin α2 and β1 were acquired from Santa Cruz Biotechnology Inc (Santa Cruz, CA, USA). Antibodies against p-FAK (Tyr 407) were from BioSource Quality Controlled Biochemicals, Inc. (Morgan Hill, CA, USA). Goat anti anti-mouse IgG H&L chain specific peroxidase conjugate and anti-rabbit IgG conjugated to horseradish peroxidase were from Calbiochem (Barcelona, Spain). Phalloidin-FITC, PI3K inhibitor (Ly294002), p-FAK (Tyr 576, Tyr 861), and type I collagen were all from Sigma (St Louis, MO, USA). Antibodies against p-FAK (Tyr 577) were from Thermo Fisher Scientific (Waltham, MA, USA). All other reagents were of the highest grade of purity available.

Total proteins from cells/tissues were extracted using RIPA buffer supplemented with protease inhibitor cocktail and phosphatase inhibitors at indicated time points. Samples were subjected to SDS-polyacrylamide gel electrophoresis and transferred to methanol-pretreated PVDF membranes (Millipore Corp., Bedford, MA, USA). Primary antibodies used in this study included p-AMPK (Thr(P)-172), total AMPK (Genetex, Irvine, CA), FAS, p-PPARγ, p-C/EBPα, AP2, p-C/EBPβ, CDK2, and Cyclin A (Cell signaling, Beverly, MA). The loading control: β-actin and secondary-HRP-conjugated mouse as well as rabbit antibodies were obtained from Santa Cruz Biotechnology (Dallas, TX). Protein bands were detected with SuperSignal West Pico Chemiluminescent Substrate (ThermoFisher Scientific, Grand Island, NY) by autoradiography.

The following antibodies were used for immunoblotting, immunofluorescence, or flow cytometry: SSRP1 (609702; Biolegend, San Diego, CA), SPT16 (607002; Biolegend), HRAS (sc-520; Santa Cruz, Dallas TX), p53 (ab27696, Abcam, Cambridge, United Kingdom), p21 (sc-6246; Santa Cruz), γH2AX (9718; Cell Signaling, Danvers, MA), RPA70 (2267; Cell Signaling), MCM7 (sc-9966; Santa Cruz), MCM4 (ab4459; Abcam), phospho-(Ser/Thr) ATM/ATR substrate antibody (2851; Cell Signaling), Chk1 (2360, Cell Signaling), p-Chk1-Ser317 (2344; Cell Signaling), p-Chk1-Ser296 (90178; Cell Signaling), cyclin A (4656; Cell Signaling), PCNA (2586; Cell Signaling), ssDNA (MAB3868; MilliporeSigma), Cross-Adsorbed Secondary Antibody, Alexa Fluor 488 (A-11008; Thermo Fisher Scientific, Waltham, MA), and β-actin (A3854, Sigma-Aldrich). Where indicated, cells were treated with 1 or 2.5 mM HU (H8627; Sigma-Aldrich). Cell cycle analysis was done using propidium iodide or Hoechst 33342 stain. CBL0137 (1 μM; Incuron, Buffalo, NY, USA) was used as a positive control for p53 activation and p21 induction.