Additional file 1 of GREM1/PPP2R3A expression in heterogeneous fibroblasts initiates pulmonary fibrosis
Creators: name: Shi, Xiaoni
name: Wang, Jing
name: Zhang, Xinxin
name: Yang, Shaoqi
name: Luo, Wei
name: Wang, Sha
name: Huang, Jie
name: Chen, Mengling
name: Cheng, Yusi
name: Chao, Jie
Publisher: name: figshare
Year: 2022
Subjects: subject: Biochemistry
subject: Medicine
subject: Cell Biology
subject: Genetics
subject: FOS: Biological sciences
subject: Pharmacology
subject: Biotechnology
subject: Immunology
subject: FOS: Clinical medicine
subject: Biological Sciences not elsewhere classified

description: Additional File 1: Fig. S1. Classification of fibroblasts using single-cell transcriptome sequencing. A Markers of myofibroblast differentiation. B GO enrichment analysis of the molecular functions of the top 50 genes in Cluster 6. C GO enrichment analysis of biological processes related to the top 50 genes in Cluster 6. Fig. S2. Expression of grem1 in saline- and silica-treated mice at 56 days determined by scRNA-seq and spatial transcriptome sequencing. A The expression of grem1 in inflammatory-proliferative fibroblasts in the silica group at 56 days was higher than that in the normal saline group but lower than that at 7 days. B The spatial localization of grem1 expression in the silica group was greater than that in the normal saline group but showed a decreasing trend compared with that at 7 days. Fig. S3. Optimal concentration of TGF-β1 for cell treatment. A A representative WB showed that a TGF-β1 concentration of 5 ng/ml yielded the highest expression of FN1, COL1, and α-SMA. B The statistical analysis of three experiments showed that the cells treated with 5 ng/ml TGF-β1 exhibited the highest expression of FN1. C The statistical analysis of three experiments showed that the cells treated with 5 ng/ml TGF-β1 exhibited the highest expression of COL1. D The statistical analysis of three experiments showed that the cells treated with 5 ng/ml TGF-β1 exhibited the highest expression of α-SMA. Fig. S4. Exploration of the downstream targets of GREM1 and verification of the Grem1 knockdown efficiency. A KEGG analysis showing that GREM1 is related to BMP and PP2A in the TGF-β signaling pathway. Related research on PP2A is lacking. B The WB results show that among the three siRNAs, siRNA-Grem1-485 exhibited the highest knockdown efficiency. C The qRT–PCR results showed that among the three siRNAs, siRNA-Grem1-485 was the most efficient, achieving approximately 70% knockdown. D The tissue immunofluorescence results suggest that PPP2R3A colocalizes with GREM1 in fibroblasts. Fig. S5. Cells were treated with TGF-β1 to construct a cell model, and TGF-β1 increased cell viability and migration. A A representative WB shows that the expression of the fibrosis-related markers FN1, COL1, and α-SMA increased in a time-dependent manner in the cells treated with the optimal TGF-β1 concentration of 5 ng/ml. B *p&lt;0.05 indicates that the increase in FN1 expression from 0 h to the indicated time point was significant. C *p&lt;0.05 indicates that the increase in COL1 expression from 0 h to the indicated time point was significant. D *p&lt;0.05 indicates that the increase in α-SMA expression from 0 h to the indicated time point was significant. E The CCK-8 assay results show that TGF-β1 treatment increased the cell viability in a time-dependent manner to a peak at 72 h. *p&lt;0.05 indicates that the difference in cell viability between 0 h and the indicated time point was significant. F The results of the wound healing experiment show that TGF-β1 treatment increased cell migration. G *p&lt;0.05 indicates that the difference in cell migration between the TGF-β1 treatment group and the control group was significant. Fig. S6. Verification of the Ppp2r3a knockdown efficiency and GREM1 regulation of PPP2R3A through downstream signaling pathways. A The qRT–PCR results show that among the three siRNAs, siRNA-Ppp2r3a-1174 exhibited the highest knockdown efficiency of approximately 70%. B The WB results show that among the three siRNAs, siRNA-Ppp2r3a-1174 exhibited the highest knockdown efficiency. C The statistical analysis of three experiments showed that siRNA-Ppp2r3a-1174 yielded the highest knockdown efficiency. D GO analysis of the signaling pathways enriched with GREM1, PPP2R3A, FN1 and p53. These results and those included in Table 2 indicate that GREM1 mainly regulates PPP2R3A through the Wnt signaling pathway.