Male Sprague-Dawley rats (200-220 g) were purchased from Weitonglihua Company (Beijing, China) and maintained in a pathogen-free facility. After one week of adaptive feeding, a total of 30 rats were randomly divided into 3 groups (n = 10 rats/group): a control group; bleomycin (BLM) group; and BLM + sh-ZFAS1 group. Rats in the BLM group were administered 5 mg/kg BLM (Nippon Kayaku, Japan) dissolved in phosphate buffered saline (PBS) and administered to the rats intratracheally to establish the PF model. Rats in the control group were treated with 0.05 mL PBS. Rats in the BLM + sh-ZFAS1 group were injected intraperitoneally with 30 uL lncRNA ZFAS1 shRNA adeno-associated virus 5 (Vigene Biosciences, USA) for 3 weeks prior to an injection of 5 mg/kg BLM sulfate.

To simulate the animal model of diabetic cardiomyopathy, male db/+ mice and db/db mice (age, 7 weeks, weight, 24 g) were fed a normal diet for 4 weeks and kept at 24 under a 14-h light/8-h dark cycle. The animals were purchased from the Model Animal Research Center of Nanjing University (Nanjing, China). Diabetic mice were intracoronarily administered equal volumes (80 ul) of adenoviruses Ad-ZFAS1, Ad-sh-ZFAS1, Ad-CCND2, Ad-sh-CCND2 or Ad-NC.33 miR-150-5p mimics and mimic control (NC) were injected into the tail vein of mice (50 ug/kg) every 15 days for 12 weeks. Db/db mice were treated with or without ferrostatin-1 (Fer-1, ferroptosis inhibitor; Sigma-Aldrich, 5 mg/kg) for an additional 12 weeks.

For in vivo experiments, the eyes in each group (n = 6) were enucleated carefully and processed for indirect immunofluorescence in whole-mount or cross-section as previously described. For cryosections, the eyes (n = 3 retinae from 3 mice) were fixed in 4% PFA at room temperature for 15 min. The frozen samples were then sliced transversely (6 um) at -20. For retinal flat-mounts, the eyes (n = 3 eyes from 3 mice) were fixed in 4% PFA at room temperature for 15 min, and the retinae were dissected out as cups. Both cryosections and retinal cups were blocked with PBS containing 0.5% Triton-X100 and 5% BSA at 4 overnight and included with the anti-CD31 and anti-GPX4 (1:100, ab125066, Abcam) primary antibodies.

To simulate the animal model of diabetic cardiomyopathy, male db/+ mice and db/db mice (age, 7 weeks, weight, 24 g) were fed a normal diet for 4 weeks and kept at 24 under a 14-h light/8-h dark cycle. The animals were purchased from the Model Animal Research Center of Nanjing University (Nanjing, China). Diabetic mice were intracoronarily administered equal volumes (80 ul) of adenoviruses Ad-ZFAS1, Ad-sh-ZFAS1, Ad-CCND2, Ad-sh-CCND2 or Ad-NC.33 miR-150-5p mimics and mimic control (NC) were injected into the tail vein of mice (50 ug/kg) every 15 days for 12 weeks. Db/db mice were treated with or without ferrostatin-1 (Fer-1, ferroptosis inhibitor; Sigma-Aldrich, 5 mg/kg) for an additional 12 weeks.