Zb 2301

1-CP-U, synthesized according to the reported procedure (5 ), was generously provided by Ms. Ning Qizhi, who originally synthesized the agent. The chemical structure of 1-CP-U is demonstrated in Fig. 1. A total of 1.0 g of 1-CP-U crystal was weighed, totally dissolved it in ultrapure water facilitated by a 0.25 mol/l hydrochloric acid solution, and then the pH was adjusted to 4.0 by adding 0.25 mol/l sodium hydroxide solution. Calculating the concentration of the stock solution as 63.39 mM, it was diluted to the required concentrations in conditional medium and then stored at 4°C. The antibodies were as follows: Polyclonal rabbit anti-human Bax, MMP-2 and MMP-9, monoclonal mouse anti-human Bcl-2, (Wuhan Boster Biological Technology., Ltd.; A0315-2, BA0569, BA0573 and BM0200, respectively), and β-actin was purchased from Zhongshan Golden Bridge Biotechnology Co., Ltd., (Beijing, China; TA-09). The goat anti-mouse or anti-rabbit secondary antibodies were from Zhongshan Golden Bridge Biotechnology (ZB-2305 and ZB-2301).

Collection and lysis of PDLSCs was conducted using RIPA Lysis Buffer which contains 1% protease inhibitor cocktail (Solarbio). Protein concentration was determined by a BCA kit (Thermo) and a total of 30 μg of protein was used for western blot analysis. The primary antibodies against RUNX2 (CST, #12556), GDF5(Abcam, RRID: ab93855), p38 MAPK(Affinity, Cat#AF6456), phosphorylated p38 MAPK (Affinity, Cat#AF4001), JNK(Affinity, Cat#AF6318), phosphorylated JNK(Affinity, Cat#AF3318), ERK(Affinity, Cat#AF0155), phosphorylated ERK(Affinity, Cat#AF1015), and β-ACTIN (Abcam, RRID: ab8226) diluted at 1:1,000 overnight at 4°C. Three washes were done using TBST. Afterwards, incubation of the membranes was done with the anti-rabbit and anti-mouse secondary antibodies (ZB-2301 and ZB-2305, Zhongshan Golden Bridge Biotechnology, Beijing, China) which is diluted at 1:10,000 at room temperature for 1 h. Visualization of the bands was done by enhanced chemiluminescence using the Bio-Rad system for detection (ChemiDocTM MP Imaging System, USA). Intensity of the bands was measured using ImageJ. β-ACTIN internal control was used to ensure equal protein loading.

Whole protein lysates of liver tissue were extracted using RIPA buffer (25 mM Tris-HCl (pH 7.6), 150 mM NaCl, 1% NP-40, 1% sodium deoxycholate, and 0.1% SDS, Thermo Fisher Scientific) supplemented with 1% protease inhibitor cocktail and 1% phenylmethylsulfonyl fluoride. Protein concentrations were measured using a BCA Protein Assay Kit. Aliquots containing 50 μg of protein were loaded onto an 8% SDS-PAGE gel, transblotted onto a polyvinylidene difluoride (PVDF) membrane (Bio-Rad Laboratories, USA), blocked for 1 h at room temperature with 5% bovine serum albumin in TBS buffer, and subsequently incubated overnight with respective primary antibodies against p65, p38, ERK, caspase-9, caspase-3, and ß-actin (internal control). The membrane was then incubated with secondary HRP conjugated antibodies (ZB 2301, Zhongshan Golden Bridge Biotechnology Co. Ltd., China). The bound complexes were detected with a chemiluminescence solution (ECL) purchased from Millipore (#WBKLS0050, Merck Millipore, USA) according to the manufacturer's instructions. Chemiluminescence was imaged on a LAS-3000 system (FUJIHILM, Tokyo, Japan). The immunoblot bands were quantified using densitometric analysis, and the ratio of each protein to ß-actin was calculated and normalized to the values of the mice fed the normal chow (set as 1).

For western blotting, 120 oocytes were lysed in 2×SDS sample buffer, boiled for 5 min at 100°C and then subjected to 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The separated proteins were transferred to a polyvinylidene difluoride (PVDF) membrane. Membrane was blocked in Tris-buffered saline (TBS) containing 5% BSA and 0.1% Tween-20 for 2 h and then incubated with rabbit anti-SIRT1 antibody (1:1000) (9475; Cell Signaling Technology, Inc.) and mouse anti-β-actin antibody (1:1000) (BE0021; Easybio Technology, Beijing). After multiple washes in TBS containing 0.1% Tween-20 and incubation with horseradish peroxidase (HRP)-conjugated anti-rabbit IgG (1:1000) (ZB-2301; Zhongshan Golden Bridge Biotechnology, Beijing) and horseradish peroxidase conjugated anti-mouse IgG (1:1000) (ZB-2305; Zhongshan Golden Bridge Biotechnology, Beijing), respectively, finally, the membranes were washed 3 times in TBS containing 0.1% Tween-20 and visualized using Bio-Rad ChemiDoc XRS+.