Bicinchoninic acid method

The cultured cells were harvested to extract total proteins with RIPA lysis (Thermo Scientific, USA, and their concentration was detected using bicinchoninic acid method (Thermo Scientific, USA), then adjusted to 4μg/μL. Following separating with 12% SDS-PAGE, the proteins were transferred to a polyvinylidene difluoride membrane. Subsequently, the membrane was dyed in Ponceau S working solution, soaked in PBST for 5 min and washed, sealed with 5% non-fat milk powder for 2 h, and incubated overnight at 4° C along with primary antibodies against CD9 and CD63, MMP-14 (1: 1000, ABCM, USA). After washing the membrane to remove the primary antibodies, horseradish peroxidase labeled goat anti-rabbit (Abcam, USA) secondary antibody (1: 5000) was added for another 1-h incubation (37° C). Afterwards, the membrane was washed 3 times with PBS for 5 min each, and the excess liquid was dried with a filter paper. The development was performed with enhanced chemiluminescence (ECL) in a darkroom. Gray values of luminescent protein bands were measured with Quantity One. Relative expression of target proteins = gray value of target protein band /gray value of β-Actin protein band.

Cells were lysed in radioimmunoprecipitation assay buffer (Thermo Scientific) supplemented with 1% protease/phosphatase inhibitor cocktail (Cell Signaling Technology, Inc., Danvers, MA, USA). Protein concentrations were measured using the bicinchoninic acid method (Thermo Scientific). Cell proteins were separated on 4–12% Bis-Tris gels (Life Technologies), transferred to polyvinylidene difluoride membranes (Nippon Genetics, Tokyo, Japan), and immunoblotted as previously described^{60 (link)}. The primary antibodies are available in Supplementary Table S9.

Western blot was performed in cultured cells as indicated. The cells were prepared from cell lines with RIPA lysis buffer kit (Boster Biological Tech, Wuhan, China), and the total protein was quantified using the bicinchoninic acid method (Thermo Fisher Scientific, Waltham, MA, USA). Whole-cell proteins (25 μg) were separated using 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis. Subsequently, the gels were transferred to polyvinylidene fluoride membranes (Beyotime, Shanghai, China). Then, the membranes were blocked with 5% skim milk in TBST for 1 h at room temperature. After that, the membranes were incubated with the primary antibodies: GAPDH (1:6,000; Proteintech, USA), Ki67 (1:1,000; Abcam, MA, USA), Bax (1:1,000; Cell Signaling, Germany), Bcl2 (1:3,000; Proteintech, USA), cleaved caspase3 (1:1,000; Cell Signaling, Germany), caspase3 (1:1,000; Cell Signaling, Germany), and ATRX (1:2000; Proteintech, USA) overnight at 4°C. After washing with TBST buffer three times, membranes were reacted with the secondary goat anti–rabbit/mouse antibody conjugated with horseradish peroxidase for 1 h at room temperature. Signals were measured using enhanced chemiluminescence) kit (MD Millipore, Germany) in the Bio-Rad ChemiDocXRS Imaging system (Bio-Rad, Hercules, CA, USA).

After insulin stimulation or mitochondrial isolation, samples were tip sonicated in 2% SDS-RIPA. Insoluble material was removed by centrifugation at 21,000 g × 10 min. Protein concentration was determined by bicinchoninic acid method (Thermo Scientific). 10 μg of protein was resolved by SDS-PAGE and transferred to PDVF membranes. Membranes were blocked in Tris-buffered saline (TBS) 4 % skim milk for 30 min at room temperature, followed by primary antibody incubation (detailed antibody is provided in “Key Resource Table”). Membranes were washed in TBS 0.1% tween (TBS-T) and incubated with appropriate secondary antibodies (IRDye700- or IRDye800-conjugated) in TBS-T 2% skim milk for 45 min at room temperature. Images were obtained by using 700- or 800-nm channels using Odyssey IR imager. Densitometry analysis of immunoblots was performed using Image Studio Lite (version 5.2). Uncropped Western blots are provided in Supplementary Material.

Tissues or cells were homogenized in RIPA buffer containing 0.2% SDS, 1 mM EDTA, 1% NP-40, 0.5% sodium deoxycholate, 1 mM sodium orthovanadate, 1 mM sodium fluoride, 1 mM PMSF and EDTA-free mini-complete protease inhibitor cocktail tablet (Roche). The protein concentrations of the extracts were determined using the bicinchoninic acid method (Thermo Scientific). Proteins were separated in 10% or 12% Bis/Trisacrylamide gels (BioRad) and transferred to nitrocellulose membranes. Blocking was achieved with 5% (w/v) milk in PBS. The primary antibodies to Thap1 were rabbit polyclonal (Proteintech) used at 1:2000 dilution, mouse monoclonal 3H3 clone (Santa Cruz) used at 1:1000, and mouse monoclonal NeuroMab clone N325B/65 (UC Davis/NIH NeuroMab Facility) at 1:5 dilution. Additionally, monoclonal mouse anti-V5 (Invitrogen) was used at 1:2000. TorA was detected with ab34540 rabbit polyclonal (Abcam). All primary antibodies were incubated in in PBS-Tween 5% milk for 2 hours at room temperature or overnight at 4°C. Membranes were then washed in TBS-Tween. Secondary antibodies anti-rabbit IgG–HRP (Vector Labs) or anti-mouse IgG-HRP (Vector Labs) were used (1:10.000) in PBS-Tween 5% milk for 1 hour at room temperature. Immunoreactive proteins were visualized using Enhanced Chemiluminiscence (ECL) on a Fujifilm LAS4000 imaging device.

Protein samples were prepared from T-HESCs using radioimmunoprecipitation assay (RIPA) buffer. Cell lysates were kept on ice for 30 mins, followed by sonication and centrifugation. Supernatants containing soluble proteins were quantified using bicinchoninic acid method (Thermo Fisher Scientific). Approximately 15 μg of proteins were loaded onto 12% Mini-PROTEAN TGX Precast Gels (Bio-Rad). After electrophoresis, proteins were transferred to polyvinylidene difluoride (PVDF) membranes by using Bio-Rad Trans-Blot Turbo Transfer System. The PVDF membranes were then blocked with 5% non-fat milk and incubated with rabbit anti-COL-1 (1:1000; ab34710; Abcam), rabbit anti- connective tissue growth factor (CTGF; 1:1000; ab6992; Abcam), rabbit anti-ACTA2 (1:4000; #19245; Cell Signaling Technology), mouse anti-ITGA1 (1:250; sc-271034; Santa Cruz) and rabbit anti-GAPDH (1:1000; # 2118; Cell Signaling Technology) overnight at 4°C. Then, membranes were washed and incubated with horseradish peroxidase (HRP)-conjugated antibody (1:20,000) for 1 h at room temperature. Following antibody incubation and extensive washes, Immobilon Western Chemiluminescent HRP Substrate (EMD Millipore) was added to the membranes for signal detection. Images were developed using a Bio-Rad ChemiDoc imaging system.