Additional file 3 of Haem relieves hyperoxia-mediated inhibition of HMEC-1 cell proliferation, migration and angiogenesis by inhibiting BACH1 expression

Additional file 3 Original Western blot images. Fig. S1 Effect of haem on HRMEC cell proliferation. a, Diagram showing the effect of different concentrations of haem on HRMEC- cell proliferation as detected by the CCK-8 method. All data are presented as the means ± SD, and the experiment was repeated three times (n = 3). One-way ANOVA or multivariate ANOVA was performed. b, Diagram showing the effect of different incubation times of 20 μM haem on HRMEC cell proliferation as detected by the CCK-8 method. All data are presented as the mean ± SD, and the experiment was repeated three times (n = 3). Multivariate ANOVA with repeated measurements test was performed. c, Images representative of the proliferative activity of HRMEC cells as measured by the EdU assay. Under a laser confocal microscope, proliferating cells were stained green (488), and nuclei were stained blue (DAPI). The arrow indicates a representative HRMEC cell with proliferative activity (scale bar = 200 μm). d-f, Quantitative analysis of the total number of cells, the number of cells with proliferative activity and the percentage of cells with proliferative activity among HRMEC cells subjected to different treatments. All data are presented as the means ± SD, and the experiment was repeated three times (n = 3). One-way ANOVA or multivariate ANOVA was performed. Fig. S2 Effect of haem on the angiogenesis of HRMEC cells. a, Images of the capillary-like tube formation of HRMEC cells stained green (488) after 48 h of culture under different conditions. Under hyperoxic conditions alone, the vascular network was fragmented and disordered. Upon the addition of 20 μM haem, the vascular network was more complete and denser (scale bar = 200 μM). b-d, Quantitative analysis of the number of junctions, length and area of capillary-like tubes formed by HRMEC cells. All data are presented as the means ± SD, and the experiment was repeated three times (n = 3). One-way ANOVA or multivariate ANOVA was performed.附加文件3：原始蛋白质印迹（Western blot）图像。 补充图S1 血红素（haem）对HRMEC（人视网膜微血管内皮细胞，Human Retinal Microvascular Endothelial Cells）细胞增殖的影响。 a. 采用CCK-8法检测不同浓度血红素对HRMEC细胞增殖影响的示意图。所有数据以平均值±标准差（SD）表示，实验重复三次（n=3），采用单因素方差分析（One-way ANOVA）或多因素方差分析进行统计检验。 b. 采用CCK-8法检测20 μM血红素不同孵育时长对HRMEC细胞增殖影响的示意图。所有数据以平均值±标准差（SD）表示，实验重复三次（n=3），采用重复测量多因素方差分析进行统计检验。 c. 采用EdU（5-乙炔基-2'-脱氧尿苷）法检测HRMEC细胞增殖活性的代表性图像。在激光共聚焦显微镜下，增殖细胞被标记为绿色（488通道），细胞核经DAPI（4',6-二脒基-2-苯基吲哚）染色呈蓝色。箭头指示具有增殖活性的代表性HRMEC细胞（标尺=200 μm）。 d~f. 对不同处理条件下HRMEC细胞的总细胞数、增殖活性细胞数及增殖活性细胞占比进行定量分析。所有数据以平均值±标准差（SD）表示，实验重复三次（n=3），采用单因素方差分析或多因素方差分析进行统计检验。 补充图S2 血红素对HRMEC细胞血管生成的影响。 a. 不同条件下培养48 h后，HRMEC细胞形成的毛细血管样管状结构的绿色（488通道）染色图像。仅在高氧条件下，血管网络呈碎片化且排列紊乱；添加20 μM血红素后，血管网络更完整且更为致密（标尺=200 μm）。 b~d. 对HRMEC细胞形成的毛细血管样管状结构的连接数、长度及面积进行定量分析。所有数据以平均值±标准差（SD）表示，实验重复三次（n=3），采用单因素方差分析或多因素方差分析进行统计检验。