Cox 2

Mouse heart tissue/cells were lysed for 30 min with RIPA buffer (Beyotime, China) supplemented with protease and phosphatase inhibitors (Beyotime, China) on ice. The lysate was centrifuged at 12 000 rpm for 15 min at 4 °C, and the protein concentrations of the supernatant were analyzed with a BCA kit (Beyotime, China). Each sample (20 µg of protein) was separated by SDS‒PAGE gels and transferred to PVDF membranes. The membranes were immersed in 5% milk in TBST buffer for 1 h at room temperature, followed by incubation with primary antibodies against β‐actin (CST, 1:1000), bax (Abcam, 1:1000), caspase3 (CST, 1:1000), caspase 8 (CST, 1:1000), Cyt‐c (Proteintech, 1:1000), GPx‐4 (Abcam, 1:1000), Bcl‐2 (Affinity, 1:1000), TGF‐β (CST, 1:1000), COX‐2 (Affinity, 1:1000), VEGF A (CST, 1:1000), FAK (CST, 1:1000), ERK1/2 (CST, 1:1000), p38 (CST, 1:1000), transferrin receptor (Abcam, 1:1000), and ferroportin (Novus, 1:1000) at 4 °C overnight. Then, the membranes were washed three times with TBST, followed by incubation with secondary antibodies (1:10 000) for 1 h at room temperature. The bands were visualized by using a gel documentation system (Bio‐Rad, USA) and quantified by ImageJ software.

HT-29 cells were grown in 6-well plates, treated with different drugs, and incubated for 48 h. Total cellular proteins were extracted with RIPA rapid lysate, and protein concentrations were quantified using the BCA protein analysis kit. The proteins in the cells were separated by SDS-PAGE separation gel and transferred to PVDF membranes, which were closed in 5% skim milk for 1 h, washed three times in TBST, and incubated with COX-2 (1:1000; Affinity), NF-κB monoclonal antibody (1:1000; manufacturer) monoclonal antibody. And GAPDH was used as a loading control. Then, the immunoblots were incubated with HPR-coupled goat anti-rabbit secondary antibodies (Sollerbauer, SE134) for 1 h at 37°C, stained with enhanced ECL chemiluminescent substrate, and visualized by Tanon 5200 chemiluminescent imaging system. The gray density of each protein band was normalized to the gray density of GAPDH. Each assay was repeated in triplicate.