Gtx100064

We separated protein samples with SDS-PAGE gels. After that, it was transferred to PVDF membranes (Millipore, USA). Then blocked the membranes with non-fat milk (5%) which was diluted with TBST, and incubated the membranes with primary antibodies overnight at 4 °C. Second day, after three times 10-min TBST washes, we incubated the membranes with the appropriate secondary antibodies (1:5000) at 27 °C for 2 h. Lastly, after the three times of 5-min TBST washes, detect the protein bands with a BioRad imaging system (Bio-Rad, USA). The primary antibodies were listed as below: SIRT3(1:1000, C73E3, Cell Signaling), Ac-lys (1:1000,9441S, Cell Signaling), phospho-AMPK (1:1000, D4D6D, Cell Signaling), AMPK (1:1000, 2532S, Cell Signaling), Mfn2 (1:1000, D2D10, Cell Signaling), Mfn1 (1:1000, A9880, ABclonal), Bax (1:1000, gtx32465, Gene Tex), Bcl2 (1:1000, gtx100064, Gene Tex),β-actin(1:3000, wh096194, Wanleibio).

The selected samples were collected and homogenized in lysis buffer containing 1% protease inhibitor. Protein concentrations were measured using a BCA Protein Assay kit (Thermo Scientific). Protein samples were separated on sodium dodecyl sulphate‐polyacrylamide gel electrophoresis gels and transferred to polyvinylidene fluoride membranes (Millipore). Following blocking in 5% skim milk solution in tris‐buffered saline with Tween 20 (TBST), incubated the membranes with primary antibody at 4°C for 12 hours. Next, incubated the membranes with the corresponding horseradish peroxidase‐conjugated secondary antibodies (1:5000, WH112425, ABclonal) for 2 hours, followed by three 5 minutes TBST washes. Protein bands were visualized using the BioRad imaging system (Bio‐Rad). The following primary antibodies were used: anti‐p‐Smad3 (1:1000, ab52903, Abcam), anti‐Smad3 (1:1000, ab28379, Abcam), anti‐ATF4 (1:1000, D4B8, Cell Signaling), anti‐CHOP (1:1000, L63F7, Cell Signaling), anti‐VDAC (1:1000, D73D12, Cell Signaling), anti‐PGC‐1α (1:1000, 4C1.3, Calbiochem), anti‐Bax (1:1000, gtx32465, Gene Tex), anti‐Bcl2 (1:1000, gtx100064, Gene Tex), anti‐cytochrome c (1:1000, wh118104, Wanleibio), anti‐β‐actin (1:3000, wh096194, Wanleibio), anti‐GAPDH (1:3000, LM16989, Proteintech).

After NaF treatment, total protein from hESCs was isolated using the mirVana™ miRNA Isolation kit (Life technologies). Protein lysates (50 μg/sample) were analyzed by western blot using primary antibodies, including mouse anti-human JNK (SC-7345, Santa Cruz Biotechnology, Santa Cruz, CA), mouse anti-human p-JNK (SC-293136, Santa Cruz Biotechnology), rabbit anti-human ERK (GTX59618, Genetex, Irvine, CA), rabbit anti-human p-ERK (2219–1, Epitomics, Burlingame, CA), rabbit anti-human BCL-2 (GTX100064, Genetex), rabbit anti-human BAX (GTX109683, Genetex) and mouse anti-human GAPDH (TA08, ZSGB-BIO, Beijing, China). The horseradish peroxidase (HRP)–conjugated donkey anti-mouse IgG (ZB5305, ZSGB-BIO) or donkey anti-rabbit IgG (ZB5301, ZSGB-BIO) were used as secondary antibodies. SuperSignal West Pico Trial Kit (Thermo Scientific, Rockford, IL) was applied for protein detection. The intensity of individual bands was quantified using the ImageJ densitometry software.