Fig 1: Elevated FSTL3 expression in gastric cancer is linked to poor patient prognosis. (A) Profiling of FSTL3 expression in 33 cancers within the TCGA database. (B) Analysis of FSTL3 expression in non-cancerous and cancerous gastric tissues using the GEO clinical dataset GSE33335. (C) Kaplan-Meier analysis of gastric cancer patient overall survival after classification into high (red) and low (blue) FSTL3 expression groups (n=175). Significance indicated as follows: *P<0.05, **P<0.01, ***P<0.001.
Fig 2: FSTL3 mRNA is regulated by miR-486-5p levels. (A) Schematic depiction of the predicted binding site for miR-486-5p in the FSTL3 mRNA. (B) Analysis of miR-486-5p levels in gastric cancer tumors vs. normal tissues in the STAD database in TCGA. (C) Correlative analysis of miR-486-5p and FSTL3 levels in gastric cancer tumors using the STAD database. (D) Profiling of miR-486-5p levels in normal and cancer gastric cancer cell lines using qRT-PCR. (E) Western blotting was used to analyze protein and RNA levels in gastric cancer MGC-803 cells subjected to transfection with miR-486-5p mimic or miR-486-5p inhibitor. (F) A dual luciferase assay was carried out using transfection of luciferase plasmid constructs alongside miR-486-5p mimic or miR-486-5p inhibitor. HEK 293T cells co-transfected with Luc-FSTL3-wt (WT) or Luc-FSTL3-mut (MUT) plasmids with synthetic miR-486-5p constructs. Error bars indicate +SEM; significance indicated by asterisks, *P<0.05, **P<0.01, ***P<0.001; number of experiments, n=3.
Fig 3: Differential gene expression and functional enrichment during FSTL3 overexpression. (A) Volcano map of differentially expressed genes under conditions of elevated FSTL3 levels; adjusted P<0.05. (B) The top 10 differentially expressed genes (linked to FSTL3 overexpression) in gastric cancer disease presented as a gene expression heat map. (C) Gene ontology (GO) term enrichment analysis; adjusted P<0.05. (D) Kyoto Encyclopedia of Genes and Genomes (KEGG) biochemical pathway enrichment analysis; adjusted P<0.05. (E) Protein-protein interaction (PPI) network analysis for 122 differentially expressed genes linked to gastric cancer; minimum required interaction score: 0.900. (F) Specific PPI nodes within the full network with MYOCD tool in Cytoscape.
Fig 4: FSTL3 expression promotes gastric cancer tumorigenicity in vivo. (A) Bioimaging of MGC-803-derived tumors in the flanks of Nude mice. Growth of tumors in Nude mice injected for control MGC-803 cells (NC) or cells expressing shRNA-specific for FSTL3 (sh-FSTL3). (B) Excision and viewing of subcutaneous tumors derived from Nude mice injected for control MGC-803 cells (NC) or cells expressing shRNA-specific for FSTL3 (sh-FSTL3). (C) Analysis of weight or volume of tumors derived from Nude mice injected for control MGC-803 cells (NC) or cells expressing shRNA-specific for FSTL3 (sh-FSTL3). (D) Hematoxylin-eosin (upper panels) or Ki67 immunohistochemistry (lower panels) on tissue sections from subcutaneous tumors derived from Nude mice injected for control MGC-803 cells (NC) or cells expressing shRNA-specific for FSTL3 (sh-FSTL3). (E) Analysis of FSTL3 and Ki67 using Western blotting of tumors from Nude mice injected for control MGC-803 cells (NC) or cells expressing shRNA-specific for FSTL3 (sh-FSTL3). Blotting of tumor lysates using antibodies to FSTL3 or Ki67; antibodies to ß-actin were used to check for protein loading in tumor lysates. (F) Bioluminescence imaging results of the lung metastasis frequency from Nude mice injected for control MGC-803 cells (NC) or cells expressing shRNA-specific for FSTL3 (sh-FSTL3). (G) Statistical analysis of luminescence intensity. Error bars indicate +SEM; significance indicated by asterisks, *P<0.05, **P<0.01, ***P<0.001; number of experiments, n=3.
Fig 5: Evaluating miR-486-5p specificity for FSTL3 mRNA. (A) Western blotting evaluation of FSTL3 protein levels after transfection of sh-FSTL3 and/or miR-486-5p inhibitor into MGC-803 gastric cancer cells. Monitoring ß-actin levels was used as an internal control in these blots. (B) Western blot results were quantified by using Image J software. Error bars indicate +SEM; significance indicated by asterisks, **P<0.01, ***P<0.001; number of experiments, n=3. (C) Transfection of sh-FSTL3 and/or miR-486-5p inhibitor into MGC-803 gastric cancer cells and evaluation of cell proliferation using EdU assay (24h). (D) Wounded cell monolayer closure assay after transfection of sh-FSTL3 and/or miR-486-5p inhibitor followed by imaging at 8 and 24 h post-wounding. (E) The Transwell assay for cell migration after transfection of sh-FSTL3 and/or miR-486-5p inhibitor was into MGC-803 cells. Sh-NC construct (shRNA) is a negative control for FSTL3 knockdown. (F) Quantified results of EdU, wound healing and transwell migration assays. Sh-NC was analyzed as a control.
Supplier Page from Abcam for Anti-FSTL3 antibody