Imaging analysis system

Total proteins were isolated from cells or tissues using the RIPA lysis buffer (Beyotime, China) and subjected to 10% polyacrylamide gel electrophoresis. After transferring to PVDF membrane, blocking with 5% skim milk, incubation with primary antibodies and secondary antibodies, the proteins on the membrane were finally detected by ECL kit (EpiZyme, China) and imaging analysis system (BioRad, USA). The primary antibodies used in this study were as follows: anti-GAPDH (Bioworld, 1:10000), anti-CDK2 (Bioworld, 1:1000), anti-Cyclin E1 (Bioworld, 1:1000), anti-CDK4 (Bioworld, 1:1000), anti-Cyclin D1 (Bioworld, 1:1000), anti-p53 (Bioss, 1:500), anti-p21 (Bioworld, 1:500), anti-p16 (Bioss, 1:1000), anti-ubiquitin (Santa, 1:500), anti-FTO (Bioss, 1:1000), anti-YTHDF2 (Abcam, 1:1000).

Cells were collected and lysed with lysis buffer (Thermo Fisher Scientific, Waltham, MA, USA) supplemented with phenylmethylsulfonyl fluoride (Beyotime) on ice for 30 min. The supernatant was collected after centrifugation at 13,200 rpm for 5 min. The BCA colorimetric method was used to measure the protein concentration. The samples were boiled for about 10 min. The proteins were then separated by SDS-PAGE with 10% Bis-Tris gels (Bio-Rad, Hercules, CA, USA) and transferred to PVDF membranes. Membranes were blocked in 5% skim milk in TBST for 1.5 h and incubated with primary antibodies overnight at 4 °C. And then incubated with secondary antibodies at room temperature for 1 h. Target protein bands were visualized by enhanced chemiluminescence (Thermo Fisher Scientific) and detected by an imaging analysis system (Bio-Rad). The antibodies were as follows: anti-PL (1:100; ab15554, Abcam, Cambridge, US), anti-GAPDH (1:2000; 10494-1-AP, Proteintech, NJ, USA), and anti-rabbit IgG (1:3000; 7074S, Cell Signaling Technology, Danvers, MA, USA).

Cells were withdrawn at different time intervals and collected by centrifugation at 12,000×g for 10 min. The pellets were washed and resuspended in Na₂HPO₄-citric acid buffer (50 mM, pH 6.5) and disrupted by sonication. The cell lysates were centrifuged to separate the soluble and insoluble fraction. The supernatants were used to detect the enzyme activity. Simultaneously, samples including soluble and insoluble fractions were collected and analyzed by 12% (w/v) sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE). In the cell autolysis section, the culture medium was also used to detect the enzyme activity and analyzed by SDS-PAGE. Protein bands were analyzed by density scanning with an imaging analysis system (Bio-Rad, USA). Protein concentration was determined by the BCA method.
β-Glucosidase activity was determined using pNPG as substrate. The assay mixture consisted 25 μL appropriately diluted protein sample, 475 μL 50 mM Na₂HPO₄-citric acid buffer (pH 6.5), and 5 mM pNPG. Enzyme activity was determined by monitoring absorption change at 405 nm. A unit of enzyme activity was defined as the amount of β-glucosidase required for releasing 1 μmol of pNP per minute. Reactions with heat-treated samples were used as controls. Enzyme activity was given as the averages of three separate experiments performed induplicate.