P16ink4a

NP cells were lysed with RIPA lysis buffer containing protease and phosphatase inhibitor cocktail (NCM Biotech). The following antibodies were used for western blotting: rabbit monoclonal antibodies: (GAPDH, 1:1000 dilution, #5174, CST; p62, 1:1000 dilution, ab240635, Abcam; FNDC5, 1:1000 dilution, ab174833, Abcam; p-AMPK, 1:1000 dilution, #2535, CST; AMPK, 1:1000 dilution, #5832, CST; p-mTOR, 1:1000 dilution, #5536, CST; mTOR, 1:1000 dilution, #2983, CST); rabbit polyclonal antibodies: (cleaved-caspase3 1:1000 dilution, #9661, CST; p16INK4a 1:1000 dilution, A0262, ABclonal); mouse monoclonal anti-LC3B (1:1000 dilution, #83506; CST).

Irradiated and non-irradiated cells were lysed in the RIPA buffer supplemented with 1X protease inhibitors. The protein samples (10–20 μg) were electrophoresed on 10–12% SDS-PAGE and transferred to polyvinylidene fluoride (PVDF) membranes (cat#LC2002, Novex, San Diego, CA and cat#10600029, GE Healthcare). Membranes were blocked in 3% bovine serum albumin (BSA, cat#7500804, Lampire Biological Laboratories) prepared in PBS with 0.1% Tween-20 (PBST), and immunoblotted with the following senescence and juvenescence (or longevity-promoting) markers: p21, p16^INK4a, Sirt1, and Nrf2 (cat#A1483, A0262, A0230, and A0674, respectively, ABClonal). β-actin was used as a loading control. The immunoreactive bands were visualized using Clarity enhanced chemiluminescent substrate (cat#1705060, Bio-Rad). The quantification of the western blot bands was performed using ImageJ software^{39 (link)}.