Fig. 2: Transcription profiles and verification of erastin-treated ESCs.

Primary ESCs were treated with erastin (30 µM) or DMSO for 12 h. A Lipid ROS levels were assessed using flow cytometry and C11-BODIPY. Representative data and statistical analyses from three independent experiments are shown. B Transmission electron microscopy analysis of mitochondria ultrastructure in ESCs under erastin treatment. C Heatmap displaying a subset of DEGs in ESCs treated with 30 µM erastin for 12 h (≥2‐fold, P < 0.05). GO (D) and KEGG pathway enrichment (F) analyses on DEGs in response to erastin-induced ferroptosis. E Heatmap of angiogenesis-related DEGs. G RT-qPCR analysis was used to validate the DEGs, which included angiogenic cytokines (VEGFA, IL8, ANGPTK4, ADM, and IL1A), inflammatory (IL2 and IL11) and growth factors (CLCF1 and AREG) (n = 3, compared with DMSO). H, I The ESCs were treated with erastin at different concentrations (10, 20, 30, 50, and 100 µM) for 12 h and for different time periods (0, 3, 6, 9, and 12 h) using 30 µM erastin. VEGFA and IL8 mRNA expressions were detected using RT-qPCR and statistical analysis from three independent experiments. * Compared with DMSO, # compared with 0 h. Arrowheads indicate deformed mitochondrial structures, while arrows point to normal mitochondrial structures. Comparisons were made using the two-tailed, Student’s t test (A, G), one-way ANOVA (H) and two-way ANOVA (I). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, ^#P < 0.05, ^##P < 0.01. ns not statistically significant, ESCs endometrial stromal cells, DMSO dimethyl sulphoxide, ROS reactive oxygen species, GO Gene Ontology, KEGG Kyoto Encyclopaedia of Genes and Genomes, DEGs differentially expressed genes, VEGFA vascular endothelial growth factor A, IL8 interleukin 8, ANGPTK4 angiopoietin-like protein 4, ADM adrenomedullin, IL1A interleukin 1A, CLCF1 cardiotrophin-like cytokine factor 1, AREG amphiregulin.