Additional file 1 of Single-cell transcriptomic analysis of endometriosis provides insights into fibroblast fates and immune cell heterogeneity

Additional file 1: Figure S1. Study flow chart. Single-cell RNA-sequencing. Figure S2 a-g. Enriched functions of FBs, Eps, ECs, T cells, NK cells, M cells, ast cells and neutrophils by GO and KEGG analyses. Figure S3. Single-cell RNA-seq analysis revealed distinct characterization of cell populations in endometriosis lesions, eutopic endometrium and normal endometrium. (A) Similarity analyses of each cell population in endometriosis lesions, eutopic endometrium and normal endometrium. (B) Differential expression analysis was performed comparing whole cells from endometriosis lesions, eutopic endometrium and normal endometrium. (C) Representative significantly enriched GO and KEGG processes were performed with the significantly upregulated genes in endometriosis lesions, eutopic endometrium and normal endometrium. (D) Expression of selected pathway genes were shown for each cell of endometriosis, eutopic endometrium, or normal endometrium origin. Dot size corresponded to the percentage of cells in the cluster expressing a gene, and dot color corresponded to the average expression level for the gene in the cluster. Figure S4. DEG analysis compared cells from endometriosis lesions, eutopic endometrium, and normal endometrium within ECs and Eps by heatmap. Figure S5. Immunofluorescence in cells showed the C3, C7, StAR and S100A10 positive FBs in endometriosis lesions. Figure S6. (A) Contribution of each group to each cell state on Figure 2E. The majority of state 1 was occupied by FBs from the eutopic endometrium and normal endometrium. State 2 was primarily contained by ectopic and eutopic endometrium FBs, while state 3 contained FBs from all three groups. (B) Relative contributions of FBs from13 subclusters to each group. (C) Relative contributions of FBs from three groups to each cluster, as shown by the t-SNE plot. (D)Western blot analysis reflected si-StAR transfection efficiency. (E) Scratch wound healing determined the ability of migration between si-Ctrl and si- StAR FBs. (F) Transwell assays were used to compare the effects of si- StAR on migration and invasion. (G) Proliferative ability between si-Ctrl and si- StAR FBs. (H) Representative images of flow cytometric cell cycle analysis. (I) Apoptosis of si-Ctrl and si- StAR FBs by flow cytometric. Figure S7. (A) Bar plot demonstrated the relative ratio of each subcluster to the entire T cell population. (B) Relative contributions of T cells from three groups to each cluster. (C) Contribution of each CD4+ T cells group to each cell state on Figure 6B. The majority state 1 was occupied by CD4+ T cells from eutopic endometrium and normal endometrium. State 2 contained CD4+ T cells from all three groups while half of CD4+ T cells in state 3 were from endometriosis lesions. (D) Contribution of each CD8+ T cells group to each cell state on Figure 6F. The majority of state 1 was occupied by CD8+ T cells from endometriosis lesions. Eutopic endometrium and normal endometrium and half of CD8+ T cells in state 2 were from endometriosis lesions. State 3 and state 4 contained T cells from all three groups while normal endometrium CD8+ T cells accounted for most of state 4. State 5 was mostly occupied by CD8+ T cells from eutopic and normal endometrium. (E) Bar plot demonstrated the relative ratio of each subcluster to three NK cell groups. (F) Relative contributions of T cells from three groups to each cluster. (G) Bar plot demonstrated the relative ratio of each subcluster to three M cell groups. (H) Relative contributions of M cells from three groups to each cluster. (I) Heatmap showed average expression of specific genes of M1 and M2 including CD64, CD40, CD86, CD163 and CD206. Figure S8. Functional enrichment analysis with GO and KEGG analyses of M-1, M-3, M-4, M-5, M-6, M-7, and M-9. Figure S9. The dense network and multiple cellular connection in eutopic endometrium and normal endometrium. (A) Putative signaling between differentially expressed receptors in different cell types and their ligands. Compartments represented cell types, and their preferentially expressed receptors and ligands were labeled along the outer margin. The solid line indicated that there was a significant interaction, and the dashed line indicated that it was not significant. (B) Capacity for intercellular communication between FBs and immune cells. Each line color indicated the ligands expressed by the cell population represented in the same color (labeled). The lines connected to the cell types that expressed the cognate receptors. Figure S10. (A)Heatmap of average gene expression of ESR2, PGR, StAR, CYP19A1. (B) Relative contributions of each cluster to each sample. (C) t-SNE plots of cells from each sample profiled in this study, with each cell color coded to indicate the associated cell types.