Protease inhibitor cocktail

Pairs of ovarian tissue were cultured for 3 h, and proteins were extracted using RIPA buffer containing a protease inhibitor cocktail (APExBIO, United States) and phosphatase inhibitors (Roche, Switzerland). Protein concentrations were determined by BCA protein assay (Beyotime Institute of Biotechnology, China). Equal amounts of protein lysates were separated on 12% PAGE Bis-Tris gels (Epizyme) and transferred onto nitrocellulose membranes. Proteins were visualized, and images were obtained using an Odyssey infrared imager.

The harvested cells were resuspended in a lysis buffer containing 50 mM Tris-HCl pH 8.0, 150 mM NaCl, 5% glycerol, 1× protease inhibitor cocktail (Apexbio, Houston, TX, USA), 5 mM β-mercaptoethanol (β-ME), and 1 mM phenylmethylsulfonyl fluoride (PMSF). The cell suspension was lysed by sonication and protein was extracted by 1% (w/v) DDM at 4 °C for 2 h. Cell debris was spun down by high-speed centrifugation (50,000× g) at 4 °C for 1 h. Expression levels by GFP intensity were measured by a microplate reader (excitation 485 nm, emission 528 nm). All experiments were conducted in triplicate. A two-tailed unpaired Student’s t-test was applied for comparison between truncations and WT, p < 0.001 indicated statistical significance.
For FSEC detection, the supernatant was loaded to a Superose 6 increase 10/300 GL column equilibrated with a running buffer (5 mM HEPES pH 7.6, 150 mM NaCl, 0.03% DDM, 2 mM β-ME). The flow rate was 0.5 mL/min and fractions were collected as 0.3 mL per tube. The fluorescence intensity for each fraction was measured by microplate reader (excitation 485 nm, emission 528 nm).

Cells were seeded at a density of 2.9 × 10⁶ cells per 150-mm tissue culture dish 24 h before infection with the indicated strains at an effective MOI of 1. Each plate of cells was lysed at 24 hpi (for ARF1) or 32 hpi (for RhoA) in 700 μl of 2× lysis buffer (50 mM Tris, 10 mM MgCl₂, 0.5 M NaCl, 2% Igepal [pH 7.5]) containing protease inhibitor cocktail (K1007; APExBio) and immediately clarified by centrifugation at 20,000 × g for 1 min at 4°C. Lysates were rapidly frozen in liquid nitrogen and stored at −80°C. Isolation of ARF1-GTP was performed as described previously (15 (link)), using GST-GGA1 agarose and 800 μg of cell lysate diluted to 1× lysis buffer using distilled water. RhoA-GTP was isolated using a similar protocol, except the GST-GGA1 was replaced with the GST-Rhotekin RBD agarose beads (BK036; Cytoskeleton). The amount of active GTPase was determined by dividing the ARF1-GTP or RhoA-GTP signal from the pulldown with the ARF1 or RhoA signal from the cell lysate (total GTPase).

Flag-CqSIRT1 plasmid was generated with PCR and subcloned into the pxj40-Flag vector (Invitrogen, Waltham, MA, USA) between the Xho I and BamH I restriction sites. HA-VP24, HA-VP26 and HA-VP28 plasmids were generated with PCR and subcloned into the pCMV-HA vector (Invitrogen, Waltham, MA, USA) between the EcoR I and Xho I restriction sites. Plasmid transient transfection was performed using Lipo293™ Transfection Reagent according to the manufacturer’s protocol (Beyotime Biotechnology, Shanghai, China). Whole-cell extracts were generated through direct lysis with Western and IP lysis buffer (20 mM Tris (pH 7.5), 150 mM NaCl, 1% Triton X-100) supplemented with protease inhibitor cocktail (EDTA-free, 100× in DMSO) (ApexBio Technology, Houston, TX, USA). Lysates were collected through centrifugation at 13,000× g for 10 min at 4 °C. Immunoprecipitation was carried out by incubating Magnetic Beads-Conjugated Mouse Anti Flag-Tag mAb at 4 °C with lysate for 2 h (ABclonal Technology, Wuhan, China), which were then washed three times with cold Western and IP lysis buffer and eluted with 1 × SDS loading buffer (Solarbio, Beijing, China).

Cell Counting Kit-8 (CCK-8), Total ROS Assay Kit, Crystal violet solution and N-acetyl-L-cysteine (NAC), Z-VAD-FMK and TUNEL Apoptosis Assay Kit (C1091) were purchased from Beyotime Biotechnology (Shanghai, China); Antimycin A, Rotenone, Carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP), Oligomycin, 2-Deoxyglucose (2-DG) and L-glutamine were purchased from Sigma-Aldrich; CPX was from Dibo Chemical Technology (Shanghai, China); Annexin V-FITC/PI Apoptosis Double Dye Detection Kit was obtained from BD Biosciences; the Pierce BCA^TM protein assay kit, MitoSOX^TM Red and Enhanced chemiluminescence (ECL) were from Thermo Fisher Scientific; Protease inhibitor cocktail was purchased from APExBIO, Houston, TX, USA); DNeasy Blood & Tissue Kit (Cat. No.: 69504) was from QIAGEN.

Proteins were first extracted using the radioimmunoprecipitation assay lysis solution with phenylmethylsulfonyl fluoride and protease inhibitor cocktail (APEXBIO, USA) and then measured using a bicinchoninic acid kit (Beyotime, China). SDS-polyacrylamide gel electrophoresis was used to separate the protein lysates, which were then transferred to polyvinylidene fluoride membranes. After blocking the membranes with 5% skim milk, they were incubated with specific primary antibodies overnight at 4 °C, followed by a 1-h incubation with secondary antibodies at room temperature. On the Mini-REPORT Tetra Electrophoresis System, proteins isolated on the membranes were visualized using an ECL chromogenic kit (Beyotime, China). The antibodies used in this study were as follows: FTO (Abcam; ab126605, 1:1,000), m6A (Abcam; ab286164, 1:1,000), METTL3 (Abcam; ab69325, 1:1,000), vimentin (CST; 5741T, 1:1,000), CDH1 (CST; 3195S, 1:1,000), Snail (Wanlei; WL01863, 1:1,000), IGF2BP2 (Proteintech; 11601-1-AP, 1:1,000), IGF2BP3 (Proteintech; 14642-1-AP, 1:1,000), DDIT4 (Proteintech; 10638-1-AP, 1:1,000), AKT (CST; 4685S, 1:1,000), TSC2 (Proteintech; 24601-1-AP, 1:1,000), p-AKT (CST; 13038S, 1:1,000), p-TSC-2 (Proteintech; 29000-1-AP, 1:1,000), AR (Proteintech; 22089-1-AP, 1:1,000), and GAPDH (Proteintech; HRP-60004, 1:1,000).