Ab156034

Senescent melanocytes treated with either DMSO or ABT-263 for 24 h, were subsequently depleted of Methionine by incubating the cells for 60 min in Methionine-free medium (21013-24, Gibco) without FBS. Then the cells were treated with 50 µM L-azidohomoalanine (Click-IT AHA, C10102, ThermoFisher Scientific) for 4 h, after which the cells were lysed in the following protein lysis buffer: 50 mM Tris-HCl pH8.0, 1%SDS, supplemented with protease inhibitors (cOmplete mini, 04693159001, Roche). Click-IT reaction was performed according to the manufacturer’s protocol using Biotin alkyne (B10185, ThermoFisher Scientific). The proteins were dissolved in RIPA-buffer (ab156034, Abcam) supplemented with protease inhibitors (Roche). Biotinylated proteins represent the nascent proteins. The protein samples have been immunoprecipitated using magnetic beads (Dynabeads™ Protein G for Immunoprecipitation; 10003D; Invitrogen) and an anti-Mcl-1 antibody (#94296, Cell Signalling Technology), followed by an immunoblot for Biotin (hyb-8; ab201341, Abcam). Immunoblots for Vinculin (Sigma-Aldrich) and Mcl-1 (Cell Signalling Technology) were used as controls.

Poorly differentiated thyroid cancer cell lines [B-CPAP (RRID: CVCL_0153) and KTC-1 (RRID: CVCL_6300)] and anaplastic thyroid cancer (ATC) cell lines [BHT-101 (RRID: CVCL_1085)] were kindly provided by the Stem Cell Bank of the Chinese Academy of Sciences. BCPAP and KTC-1 cells were cultured in RPMI 1640 supplemented with 10% FBS and 1% non-essential amino acids (Invitrogen, USA). BHT101 cells were cultured in DMEM supplemented with 20% FBS. All human cell lines have been authenticated using short tandem repeat profiling within this year, and all experiments were performed with mycoplasma-free cells. Small interfering RNAs (siRNAs) were obtained from Guangzhou RiboBio (China). Three siRNAs targeting the Asporin gene were designed and synthesized (siRNA2: 5’-GTGACGGTGTTCCATATCA-3’; siRNA4: 5’-GGAGTATGTGCTCCTATTA-3’; siRNA5: 5’-GTGCTATTCACGAGTTGTA-3’). At the time of transfection, cells were plated onto a 6-well plate at 60%–80% confluence. Transfection was performed with RNAiMAX (13778-150; Thermo Fisher) according to the manufacturer’s protocol. RNAiMAX reagent (7.5 μL) and siRNAs were diluted in Opti-MEM and incubated at room temperature for 15 min. The mixtures were then added to cells, giving a final concentration of siRNAs of 30 pmol. BCPAP, KTC-1, and BHT101 cells were cultured for 72 h after transfection and were subsequently lysed in RIPA buffer (ab156034; Abcam).

Tumors induced by orthotopic implantation were lysed in RIPA buffer (ab156034; Abcam) with phosphatase and protease inhibitors (5872; Cell Signaling) using gentleMACS Dissociator (Miltenyi Biotec). Protein concentration in lysates were determined by using Protein Assay Dye Reagent Concentration (5000006; Bio-Rad). Cell extracts were denatured, subjected to SDS-PAGE and transferred to membranes (Trans Blot Turbo, 1704156; Bio-Rad). After blocking with Blocking One (03953-95; Nacalai), the membranes were incubated with the following antibodies overnight at 4 °C: pSMAD3 (1:1000, 9520; Cell Signaling), SMAD3 (1:1000, 9513; Cell Signaling) and β-actin (1:4000, A1978; Sigma Aldrich). After 2 h incubation with the appropriate horseradish peroxidase-conjugated antibodies (1:10000, 7074/7076; Cell Signaling), the immune complexes were detected by chemiluminescence (34580; Thermo Fisher) on an Amersham Imager 600 (GE Healthcare).

Th total protein extracts were prepared from 10 to 15 mg of the deep-frozen heart tissue. To maintain the extract integrity and function, we used Complete Protease Inhibitor Cocktail (P8340, Sigma-Aldrich, St. Louis, MO, USA), Phosphatase Inhibitor Cocktail II (ab201113, Abcam, Waltham, MA, USA), PMSF (1 mM), EGTA (1 mM), and EDTA (1 mM). The proteins were isolated using a 1xRIPA buffer (ab156034, Abcam, Waltham, MA, USA). The concentration of the protein was determined by the Lowry method [15 (link)]. The samples (30–50 µg) were diluted in Laemmli buffer, separated by 12.5% SDS-PAGE, and transferred to a nitrocellulose membrane (Thermo Fisher Scientific, Wilmington, USA) [12 (link)]. The relative levels of the detected proteins were visualized using a C-DiGit Blot Scanner (LI-COR Biotechnology, Lincoln, Nebraska, USA) and normalized to the appropriate loading control. The antibodies used: (ab56788) anti-DRP1 (1:1000), (ab119685) anti-OPA (1:1000), (ab124773) anti-Mitofusin2 (1:1000), (ab54481) anti-PGC1a (1:1000), (cs2132) anti-Parkin (1:1000), (Affinity Biosciences, DF7742) anti-PINK1 (1:250), (Affinity Biosciences, AF5384) anti-SQSTM1/p62 (1:250), (Affinity Biosciences, DF8163) anti-BNIP3L (1:250), and (cs12741) anti-LC3A/B (1:1500). The anti-GAPDH (ab181602) and anti-tubulin (1:2000) (ab4074) antibodies were used as a loading control.

Liver samples were prepared for total triglyceride detection and quantification according to the manufacturer's manual (BioVision, Milpitas, CA). Liver total cholesterol (TC) was measured using colorimetric kit (Wako Diagnostics, Richmond, VA). Frozen liver samples (~10–20 mg) were homogenized in RIPA buffer (ab156034, Abcam, Cambridge, MA) that contained a protease inhibitor cocktail tablet (Roche Diagnosis, Mannheim, GR), and protein concentrations were quantified by Bradford assay (#500‐0205, Bio‐Rad Laboratories, Hercules, CA).

Animals were deeply anesthetized with ketamine and xylazine and thoracotomy was performed to expose the heart. Using a syringe coated with EDTA, the blood was drawn from the right ventricle and collected into EDTA-coated tubes (BD Microtainer® Tubes, K2 EDTA, BD 365974, BD Biosciences, San Jose, CA) and immediately placed on ice. Blood tubes were spun at 1,000–2,000xg for 10min at 4°C. Plasma was collected and stored at -80°C. Retinas were dissected and flash frozen in dry ice and then stored at -80°C. Retina lysates were prepared using RIPA buffer (ab156034, Abcam) and protease inhibitor (Mini EDTA-free protease inhibitor tablets, Roche, Basel, Switzerland).