Akr1c1

Total protein content of cell lines or the tumor was extracted by lysis in radioimmunoprecipitation assay buffer containing protease inhibitor cocktail and the protein phosphatase inhibitor, and the total protein concentration was quantified by the BCA (bicinchoninic acid) method. Equal amounts of protein lysates were resolved by sodium dodecyl sulfate -polyacrylamide gel electrophoresis and were transferred to polyvinylidene difluoride membranes. The membranes were blocked at 37°C for 1.5 h in 5% milk and then incubated 12-16 h at 4°C with the following primary antibodies: COX2 (Abcam, Cat# ab179800), AKR1C1 (Abcam, Cat# ab192785), FTH1 (Abcam, Cat# ab75973), GPX4 (Abcam, Cat# ab125066) and β-actin (Invitrogen, Cat# A1978). The membranes were then incubated with horseradish peroxidase-linked secondary antibodies (Cell Signaling Technology) and the signals were visualized by enhanced chemiluminescence using an Amersham Imager 600 (GE Healthcare). The intensities of the signals were quantified using the ImageJ software.

ALA (Cat# SML0415-25MG), (S)-(+)-α-Tetralol (Cat# 87649), and β-NADH phosphate disodium salt (Cat# 50020) were purchased from Sigma-Aldrich (MO, United States). Fresh ALA was dissolved in DMSO for each cell experiment (10 mM stock solution). Primary antibodies against GAPDH (Cat# ab181602), p-STAT3 (Ser727, Cat# ab32143), STAT3 (Cat# ab109085), and AKR1C1 (Cat# ab192785) were purchased from Abcam (Boston, MA, United States). The TMT10plex Isobaric Mass Tag Labelling Reagents (Cat# 90113) were obtained from Thermo Fisher Scientific (Boston, MA, United States). The Dual Luciferase Reporter Gene Assay Kit (Cat# RG028) was purchased from Beyotime (Shanghai, China). Cell Counting Kit 8 (CCK-8, Cat# ab228554) was obtained from Abcam (Boston, MA, United States).