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

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 < 0.01, ****P < 0.0001]. (C) Statistical analysis of the fibrotic area percentage in the endometrium (**P < 0.01, ****P < 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 < 0.001, ****P < 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 < 0.001, ****P < 0.0001]. (D-F) b-FGF, VEGF and IGF-1 levels in uterine tissue extracts from each group [*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 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 < 0.05, ****P < 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 < 0.01, ****P < 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 < 0.05, ***P < 0.001, ****P < 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 < 0.01, ***P < 0.001, ****P < 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 < 0.05, ***P < 0.001, n.s.: not significant] (n = 3/group). (B) The lipid formation was measured by MDA assay [n.s.: not significant, **P < 0.01, ****P < 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 < 0.05, **P < 0.01, ****P < 0.0001]. (E, F) Flow cytometry with C11-BODIPY was conducted for estimating the lipid ROS level (n = 3/group) [**P < 0.01, ***P < 0.001, n.s.: not significant]. (G) Cell viability was assessed with the Cell Counting Kit-8 [*P < 0.05, **P < 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 < 0.01, ***P < 0.001] (n = 3/group). (I) Lipid formation was evaluated with MDA assay (n = 3/group) [*P < 0.05, **P < 0.01, n.s.: not significant]. GSH (J) and GSSG (K) levels were quantified by relative assay kits [*P < 0.05, **P < 0.01, ***P < 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 < 0.01, n.s.: not significant]. (N) Cell Counting Kit-8 kit was utilized for estimating cell viability [**P < 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 < 0.01, ***P < 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.

附加文件1。 图S1。 miR-340转染的骨髓间充质干细胞（Bone Marrow Mesenchymal Stem Cells, BMSCs）鉴定。(A) 光学显微镜下观察miR-340/BMSCs的形态特征，比例尺：20 μm。(B-I) 流式细胞术检测miR-340/BMSCs的表面标志物：(B-D) 分别为FITC、PE及FITC同型对照。miR-340/BMSCs对CD45(E)、CD34呈阴性染色，对CD29(G)、CD44(H)及CD90(I)呈阳性染色。
图S2。 蛋白质免疫印迹法检测外泌体标志物：(A) 检测Calnexin、CD9、CD81、TSG101、CD63及Hsp70的表达水平。
图S3。 子宫内膜机械损伤造模及统计学分析流程。(A) 子宫内膜机械损伤后给予MB-exos或B-exos处理的示意图。(B) 基于子宫组织切片的子宫内膜厚度统计学分析（每组n=6）[**P<0.01，****P<0.0001]。(C) 子宫内膜纤维化面积百分比的统计学分析（**P<0.01，****P<0.0001；每组n=6）。MD：机械损伤（Mechanical Damage, MD）。
图S4。 细胞衰老标志物β-半乳糖苷酶与P21的检测。(A) 采用细胞衰老β-半乳糖苷酶染色试剂盒检测β-半乳糖苷酶的表达水平。(B) 采用免疫组织化学法检测P21的表达。
图S5。 铁死亡激活剂艾拉司他（Erastin）对MB-exos促进损伤子宫修复的影响。(A) 假手术组（Sham）、PBS组、MB-exos组及MB-exos+艾拉司他组子宫组织的马松三色染色代表性图像（每组n=6）。(B) 各组子宫内膜纤维化面积百分比的统计学分析[***P<0.001，****P<0.0001]（每组n=6）。(C) 基于各组子宫组织切片的子宫内膜厚度统计学分析（每组n=6）[***P<0.001，****P<0.0001]。(D-F) 各组子宫组织提取物中b-FGF、VEGF及IGF-1的水平[*P<0.05，**P<0.01，***P<0.001，****P<0.0001]（每组n=6）。
图S6。 B-exos或MB-exos可削弱RSL3或艾拉司他诱导的细胞活力抑制或脂质活性氧（reactive oxygen species, ROS）生成。(A) 光学显微镜下观察原代子宫内膜间质细胞形态。(B) 采用细胞计数试剂盒-8（Cell Counting Kit-8, CCK-8）检测各组（对照组、艾拉司他组、B-exos+艾拉司他组及MB-exos+艾拉司他组）的细胞活力[*P<0.05，****P<0.0001]（每组n=3）。(C) 采用细胞计数试剂盒-8定量各组（对照组、RSL3组、B-exos+RSL3组及MB-exos+RSL3组）的细胞活力（**P<0.01，****P<0.0001；每组n=3）。采用C11-BODIPY检测各组（对照组、艾拉司他组、B-exos+艾拉司他组及MB-exos+艾拉司他组）的脂质ROS水平(D)并进行分析(E)（*P<0.05，***P<0.001，****P<0.0001；每组n=3）。采用C11-BODIPY检测各组（对照组、RSL3组、B-exos+RSL3组及MB-exos+RSL3组）的脂质ROS水平(F)并进行分析(G)[**P<0.01，***P<0.001，****P<0.0001]（每组n=3）。
图S7。 METTL3调控RSL3诱导的子宫内膜间质细胞（Endometrial Stromal Cells, ESCs）铁死亡。敲低METTL3可抑制RSL3诱导的铁死亡性细胞死亡(A-G)：将METTL3短发夹RNA（short hairpin RNA, shRNA）稳定转染至ESCs，随后给予2.5 μM RSL3处理24 h。(A) 采用铁检测试剂盒定量Fe²+积累量[*P<0.05，***P<0.001，n.s.：无显著性差异]（每组n=3）。(B) 采用丙二醛（Malondialdehyde, MDA）检测试剂盒测定脂质过氧化产物生成量[n.s.：无显著性差异，**P<0.01，****P<0.0001]（每组n=3）。采用相应试剂盒定量谷胱甘肽（GSH, C）及氧化型谷胱甘肽（GSSG, D）水平（每组n=3）[n.s.：无显著性差异，*P<0.05，**P<0.01，****P<0.0001]。(E、F) 采用C11-BODIPY流式细胞术检测脂质ROS水平（每组n=3）[**P<0.01，***P<0.001，n.s.：无显著性差异]。(G) 采用细胞计数试剂盒-8检测细胞活力[*P<0.05，**P<0.01]（每组n=3）。过表达METTL3可增强RSL3诱导的铁死亡性细胞死亡(H-N)：将METTL3过表达质粒稳定转染至ESCs，随后给予2.5 μM RSL3处理24 h。(H) 采用铁检测试剂盒定量Fe²+积累量[n.s.：无显著性差异，**P<0.01，***P<0.001]（每组n=3）。(I) 采用MDA检测试剂盒测定脂质过氧化产物生成量（每组n=3）[*P<0.05，**P<0.01，n.s.：无显著性差异]。采用相应试剂盒定量GSH(J)及GSSG(K)水平（每组n=3）[*P<0.05，**P<0.01，***P<0.001，n.s.：无显著性差异]。(L-M) 采用C11-BODIPY流式细胞术检测脂质ROS水平（每组n=3）[**P<0.01，n.s.：无显著性差异]。(N) 采用细胞计数试剂盒-8检测细胞活力[**P<0.01]（每组n=3）。
图S8。 转染HMOX1 shRNA及METTL3 shRNA并经艾拉司他处理的ESCs中ROS水平及线粒体形态检测。(A) 采用C11-BODIPY流式细胞术定量脂质ROS水平[**P<0.01，***P<0.001]（每组n=3）。(B) 透射电子显微镜下观察线粒体形态，比例尺：1 μm。
图S9。 HMOX1 mRNA的m6A结合位点。