DOI: 10.6084/m9.figshare.26755441.v1
title: Additional file 1 of miR-340-3p-modified bone marrow mesenchymal stem cell-derived exosomes inhibit ferroptosis through METTL3-mediated m6A modification of HMOX1 to promote recovery of injured rat uterus

description: Additional file 1. Fig. S1. miR-340/BMSCs identification. (A) Morphological characteristics of miR-340/BMSCs determined by optical microscopy. Scale bars: 20 μm. (B-I) Flow cytometry determination of surface markers in miR-340/BMSCs. (B-D) Isotype controls for FITC, PE, and FITC. miR-340/BMSCs showed negative staining for CD45 (E) and CD34 (F) but positive staining for CD29 (G), CD44 (H), and CD90 (I). Fig. S2. Detection of exosome markers by western blotting assay. (A) Calnexin, CD9, CD81, TSG101, CD63, and Hsp70 expression levels were assessed. Fig. S3. Procedure for mechanical damage of the endometrium and statistical analysis. (A) Schematic representation of MB-exos or B-exos treatment following mechanical damage to the endometrium. (B) Statistical analysis of endometrial thickness based on histological sections of the uterus (n = 6/group) [**P &lt; 0.01, ****P &lt; 0.0001]. (C) Statistical analysis of the fibrotic area percentage in the endometrium (**P &lt; 0.01, ****P &lt; 0.0001; n = 6/group). MD: mechanical damage. Fig. S4. Detection of cell senescence markers β-galactosidase and P21. (A) β-galactosidase expression levels were assessed using Cell Senescence β-Galactosidase Staining Kit. (B) P21 expression were assessed by immunohistochemistry. Fig. S5. Effects of the ferroptosis activator erastin on MB-exos in promoting the injured uterus recovery. (A) Representative images of uterus tissues of the Sham, PBS, MB-exos, and MB-exos+erastin groups (n = 6/group) stained with Masson’s trichrome stain. (B) Statistical analysis of the percentage of the endometrial fibrotic area in each group [***P &lt; 0.001, ****P &lt; 0.0001] (n = 6/group). (C) Statistical analysis of endometrial thickness based on histological sections of the uterus in each group (n = 6/group) [***P &lt; 0.001, ****P &lt; 0.0001]. (D-F) b-FGF, VEGF and IGF-1 levels in uterine tissue extracts from each group [*P &lt; 0.05, **P &lt; 0.01, ***P &lt; 0.001, ****P &lt; 0.0001] (n = 6/group). Fig. S6. B-exos or MB-exos impairs the inhibition of cell viability or lipid ROS production induced by RSL3 or erastin. (A) Primary endometrial stromal cell morphology was observed by optical microscopy. (B) Cell viability was determined by the Cell Counting Kit-8 for each group (control, erastin, B-exos+erastin, and MB-exos+erastin) [*P &lt; 0.05, ****P &lt; 0.0001] (n = 3/group). (C) Cell viability was quantified with the Cell Counting Kit-8 in each group (control, RSL3, B-exos+RSL3, and MB-exos+RSL3) (**P &lt; 0.01, ****P &lt; 0.0001; n = 3/group). Lipid ROS levels were estimated by C11-BODIPY (D) and analyzed (E) in each group (control, erastin, B-exos+erastin, and MB-exos+erastin) (*P &lt; 0.05, ***P &lt; 0.001, ****P &lt; 0.0001; n = 3/group). Lipid ROS levels were detected using C11-BODIPY (F) and analyzed (G) in each group (control, RSL3, B-exos+RSL3, and MB-exos+RSL3) [**P &lt; 0.01, ***P &lt; 0.001, ****P &lt; 0.0001] (n = 3/group). Fig. S7. METTL3 regulates RSL3-induced ferroptosis of ESCs. Knockdown of METTL3 suppressed RSL3-induced ferroptotic cell death (A-G). METTL3 shRNA was stably transfected into ESCs followed 24-h RSL3 (2.5 μM) treatment. (A) Fe2+ accumulation was quantified by an iron detection assay [*P &lt; 0.05, ***P &lt; 0.001, n.s.: not significant] (n = 3/group). (B) The lipid formation was measured by MDA assay [n.s.: not significant, **P &lt; 0.01, ****P &lt; 0.0001] (n = 3/group). GSH (C) and GSSG (D) levels were quantified by relative assay kits (n = 3/group) [n.s., not significant, *P &lt; 0.05, **P &lt; 0.01, ****P &lt; 0.0001]. (E, F) Flow cytometry with C11-BODIPY was conducted for estimating the lipid ROS level (n = 3/group) [**P &lt; 0.01, ***P &lt; 0.001, n.s.: not significant]. (G) Cell viability was assessed with the Cell Counting Kit-8 [*P &lt; 0.05, **P &lt; 0.01] (n = 3/group). METTL3 overexpression enhanced RSL3-induced ferroptotic cell death (H-N). METTL3 plasmid was stably transfected into ESCs followed by 24-h RSL3 (2.5 μM) treatment. (H) Fe2+ accumulation was estimated by an iron detection assay [n.s.: not significant, **P &lt; 0.01, ***P &lt; 0.001] (n = 3/group). (I) Lipid formation was evaluated with MDA assay (n = 3/group) [*P &lt; 0.05, **P &lt; 0.01, n.s.: not significant]. GSH (J) and GSSG (K) levels were quantified by relative assay kits [*P &lt; 0.05, **P &lt; 0.01, ***P &lt; 0.001, n.s.: not significant] (n = 3/group). (L-M) Flow cytometry with C11-BODIPY was carried out for estimating the lipid ROS level (n = 3/group) [**P &lt; 0.01, n.s.: not significant]. (N) Cell Counting Kit-8 kit was utilized for estimating cell viability [**P &lt; 0.01] (n = 3/group). Fig. S8. Detection of ROS level and mitochondrial morphology in ESCs transfected with HMOX1 shRNA and METTL3 shRNA followed by erastin treatment. (A) Flow cytometry with C11-BODIPY was conducted for quantifying the lipid ROS level [**P &lt; 0.01, ***P &lt; 0.001] (n = 3/group). (B) Mitochondrial morphology observed through transmission electron microscopy. Scale bars: 1 μm. Fig. S9. m6A binding site of HMOX1 mRNA.