P21waf1

FGF21 (P6101) was obtained from Beyotime Biotechnology (Jiangsu, China), chloroquine (CQ) was obtained from Sigma-Aldrich Chemical Company (Milwaukee, WI, USA), and nicotinamide (NAM) was purchased from MedChemExpress (NJ, USA). The primary antibodies directed against Collagen II, MMP-13, p16INK4a, p21WAF1, Lamin B1, and GAPDH were purchased from Abcam (Cambridge, MA, USA), while the primary antibodies directed against Bax, Bcl-2, SQSTM1/p62, and LC3 were obtained from ProteinTech (Wuhan, China). The antibodies against cleaved caspase3 (C-caspase 3), TFEB, mTOR, Phospho-mTOR (p-mTOR), and SIRT1 were procured from Cell Signaling Technologies (Danvers, MA, USA).

Cell lysates were prepared using RIPA lysis buffer containing 25 mM Tris-HCl (pH 7.6), 150 mM NaCl, 1% NP-40, 1% sodium deoxycholate and 0.1% SDS. The lysates were centrifuged at 15,000 rpm for 10 min. The lysates were mixed with Laemmli sample buffer and boiled at 98°C for 2 min. The protein samples were run on SDS-PAGE along with protein markers for SDS-PAGE (Nacalai Tesque, 02525–35, Kyoto, Japan) and blotted onto a PVDF membrane (GE healthcare, Illinois, US). The following primary antibodies were used: Tbc1d24 (ProteinTech, 1:1000, Illinois, US; LSBio, 1:1000, Illinois, US), p21 Waf1 (Abcam, ab109199, 1:1000, Cambridge, UK), p27 Kip1 (Cell Signaling Technology, #3698, 1:1000, Massachusetts, US), Impdh (ProteinTech, 12948-1-AP, 1:100), Cpts1 (ProteinTech, 15914-1-AP, 1:100), Arf6 (Cell Biolabs, STA-407-6, 1:500, California, USA) and beta-actin (Cell Signaling Technology, #5174, 1:4000). HRP-conjugated anti-mouse IgG (Thermo Fisher Scientific, 32430) and anti-rabbit IgG (Thermo Fisher Scientific, 32460) were used as secondary antibodies at 1:1000 dilution. For detection of immunoreactive bands, Chemi-Lumi One L or Chemi-Lumi One Ultra (Nacalai Tesque) were used. Uncropped blots were shown in S1 Raw images. Densitometric analyses for blotting data were performed using Fiji [39 (link)].