Additional file 1 of Exploring new frontiers: cell surface vimentin as an emerging marker for circulating tumor cells and a promising therapeutic target in advanced gastric Cancer
Creators: name: Li, Heming
name: Zhu, Yang-Zhuangzhuang
name: Xu, Lu
name: Han, Tao
name: Luan, Jiasi
name: Li, Xin
name: Liu, Yuting
name: Wang, Zhi
name: Liu, Qiuge
name: Kong, Xiangyu
name: Zou, Chunpu
name: Su, Lin
name: Hou, Yifei
name: Chen, Xiao
name: Chen, Lujun
name: Wang, Ruoyu
name: Xu, Zihang
name: Zhao, Mingfang
Publisher: name: figshare
Year: 2024
Subjects: Biophysics
Biochemistry
Space Science
Medicine
Cell Biology
Molecular Biology
Chemical Sciences not elsewhere classified
Biological Sciences not elsewhere classified
Cancer

description: Additional file 1: Fig. S1: Representative images of HER-2 IHC staining from the same case (× 100). Fig. S2:The CSV+ GC cells in subcutaneous xenograft mouse models. BGC-823 cells (4 × 106/mouse) were injected subcutaneously into the right scapula of the nude mice. (A) The tumor volume. (B) The metastasis nodules were not observed in the lung. (C) Flow cytometry showing the number of CSV+ GC cells both in the tumor tissues and blood samples. NS = not significant. Fig. S3: m6A methylation patterns based on a cohort of 300 GC patients from the Asian Cancer Research Group (ACRG). (A) Clustered heatmap of the gene expression levels of two Eraser genes (FTO and ALKBH5) across samples, with the color bar indicating clinical information distribution based on the clustering results. (B) Distribution bar graph of ACRG subtype in EraserCluster A and EraserCluster B. (C) Survival prognostic KM curve for EraserCluster A and EraserCluster B groups, with blue and red lines indicating samples in EraserCluster A and EraserCluster B, respectively. (D) PCA plot of samples based on two Eraser genes, with blue and red dots representing the samples in EraserCluster A and EraserCluster B, respectively. (E) Distribution differences in Enrichment-Score among angiogenesis, CD8 T effector, EMT1, EMT2, EMT3, and Pan-fibroblast TGFβ between EraserCluster A and EraserCluster B. *P &lt; 0.05; *** P &lt; 0.001. Fig. S4: Both proliferation and migration abilities are inhibited in GC cells after FTO inhibitor FB23-2 treatment. (A &amp; B). The colony-formation capacity of GC cells was evaluated after treatment with FB23-2 at concentrations of 0 μM, 1 μM, and 5 μM). (C &amp; D) The migration ability of GC cells was assessed under the same FB23-2 treatment conditions. *** P &lt; 0.001. Fig. S5: Differentially expressed genes in FTO knockdown HGC-27 cells based on RNA-seq analysis. Different gene profiles after FTO knockdown were presented as Heatmap (A) and RNA-seq volcano plot (B). Fig. S6: Evaluation the role of FTO among cancers and the association of FTO level and IGF-IR expression. (A) FTO expression in 61 cancers. (B-Q) High FTO level is correlated with poor prognosis (B-G) and upregulated expression of IGF-IR in cancers (H-Q). Fig. S7: Knockdown of m6A readers YTHDF1/2/3 in GC cell lines. Effect of YTHDF1/2/3 knockdown, as verified at mRNA level in both BGC-823 (A-D) and HGC-27 cells (E-H). * P &lt; 0.05; ** P &lt; 0.01; *** P &lt; 0.001; NS = not significant. Fig. S8: GSEA showing positive enrichment of EMT process with IGF-I/IGF-IR signaling pathway-related gene sets. (A &amp; B) GSEA was performed using the JAVA program ( https://www.broadinstitute.org/gsea ) to identify the IGF-I-related gene sets via MSigDB. Fig. S9: IGF-I is a crucial regulatory factor in GC cells during the EMT process. (A) GC cells were serum-starved overnight and then treated with or without 100 ng/mL IGF-I for 48 h. Images were captured at 20× magnification. (B) Results from migration assays performed using Transwell chamber methods. (C) Western blot analysis of cell lysates for EMT markers. Fig. S10: TMT quantitative proteomics of HGC-27 cell line after IGF-I stimulation. (A) GC cells were serum-starved overnight and then treated with or without 100 ng/mL IGF-I for 48 h. Proteins were extracted for proteomics analysis. Heatmap showing the abundance of EMT genes in the control and IGF-I stimulation groups. (B &amp; C) Wheel plot showing the differentially expressed gene enrichment using GO and KEGG. (D) PPI subnetwork of differentially expressed EMT-related proteins. Fig. S11: IGF-I induced the phosphorylation of vimentin in GC cells. (A) GC cells were serum-starved, followed by treatment with 100 ng/mL IGF-I for 6, 24, or 72 hours. Phosphorylated vimentin levels (at serine residues S39, S72, S82) were measured by Western blot. (B) GC cells were serum-starved and subsequently treated with 100 ng/mL IGF-I, both alone and in combination with GSK 1838705A, for 6, 24, or 72 hours. Phosphorylated vimentin levels (S39) were assessed using Western blot assay. (C) GC cells were transiently transfected with wild type or specific S39 mutant plasmid for 48 h followed by IGF-I stimulation for 48 h, the expression levels of CSV were detected through flow cytometry. ***P &lt; 0.001; NS = not significant. Fig. S12: (A) IHC staining of Ki67 expression of tumor lesions in different groups. Bar = 2mm. (B) Representative H&amp;E-stained sections of metastatic nodules in lung tissue from the mouse model. Bar = 20 μm.