Journal: Cell Death & Disease

Article Title: Cancer associated fibroblast derived SLIT2 drives gastric cancer cell metastasis by activating NEK9

doi: 10.1038/s41419-023-05965-z

Figure Lengend Snippet: A Representative images of IHC staining for SLIT2 in adjacent nontumor and CAFs in GC tissues. Long scale, 50 μm, short scale, 20 μm. B Representative images of IF staining for SLIT2 level in Normal fibroblasts and CAFs cells. C Representative images of IF staining for SLIT2 (red) and α-SMA (green) in CAFs of GC tissues. ( D ) SLIT2 levels in serum from healthy volunteers ( n = 96) and gastric cancer patients via ELISA ( n = 96). E SLIT2 promoted GC MKN45 and AGS cell migration, ROBON treatment inhibited cell migration by in vitro transwell analysis, SLIT2, recombinant, 100 ng/ ml, 24 hours, ROBON, a soluble ROBO1 receptor antagonist. F Representative images of cytoskeleton staining with phalloidin for 40 min at room temperature for SLIT2 treatment in GC cells, Scale, 50 μm. G Left panel represents the co-culture pattern diagram in human gastric cancer cells, CAFs and CAF culture supernatant. Right panel demonstrates either CAFs or their culture supernatant can promote GC cell migration, while ROBON and SLIT2 knockdown in CAFs antagonizes cell migration by in vitro transwell analysis. ** P < 0.01 and *** P < 0.001. CAF cancer-associated fibroblasts, GC gastric cancer, ROBO1, roundabout guidance receptor 1, SLIT2Δ, SLIT2 knockdown in CAFs.

Article Snippet: CAF cancer-associated fibroblasts, GC gastric cancer, ROBO1, roundabout guidance receptor 1, SLIT2Δ, SLIT2 knockdown in CAFs.

Techniques: Immunohistochemistry, Staining, Enzyme-linked Immunosorbent Assay, Migration, In Vitro, Recombinant, Co-Culture Assay, Knockdown

Journal: Cell Death & Disease

Article Title: Cancer associated fibroblast derived SLIT2 drives gastric cancer cell metastasis by activating NEK9

doi: 10.1038/s41419-023-05965-z

Figure Lengend Snippet: A Mass-spectrometry analysis was used to predict the interacting proteins of ROBO1. B The protein domain structure diagram for ROBO1 Ig domains, three fibronectin III repeats, intracellular domain and NEK9 kinase domain were predicted by SMART online database. C Co-immunoprecipitation (Co-IP) assay demonstrated the interaction between endogenous ROBO1 and NEK9 in GC cells, and SLIT2 treatment (100 ng/ ml, 24 hours) enhanced this interaction. D The GC cell lysates co-transfected with Flag-NEK9 and HA-ROBO1 (the full-length or deletion mutants) were used to detect the interaction using Co-IP assay. E Co-IP assay demonstrating the interaction between ROBO1 and mutation kinase domain of NEK9 in GC cells. ROBO1, roundabout guidance receptor 1.

Article Snippet: CAF cancer-associated fibroblasts, GC gastric cancer, ROBO1, roundabout guidance receptor 1, SLIT2Δ, SLIT2 knockdown in CAFs.

Techniques: Mass Spectrometry, Co-Immunoprecipitation Assay, Transfection, Mutagenesis

Journal: Cell Death & Disease

Article Title: Cancer associated fibroblast derived SLIT2 drives gastric cancer cell metastasis by activating NEK9

doi: 10.1038/s41419-023-05965-z

Figure Lengend Snippet: A AGS and MKN45 cells were transfected NEK9 overexpression and mutant NEK9 (kinase-dead mutant NEK9) plasmid, and the cell migration ability was evaluated via in vitro Transwell assay. B Representative images of IF staining for NEK9 (red) and α-tubulin (green) in AGS and MKN45 cell for cell shape and lamellipodia formation, Scale, 20 μm. C Phosphoproteomics analyses with stable AGS and MKN45 NEK9-overexpression transfection was used to screen the 57 candidate effector proteins of NEK9. D and E Western blot analysis for the phosphorylation levels of TRIM28 ( D ) and CTTN ( E ) in AGS and MKN45 cells with NEK9 overexpression treatment. F NEK9 was found to phosphorylate TRIM28 and CTTN using an in vitro kinase assay. G and H Co-immunoprecipitation assay illustrating the interaction between NEK9 and TRIM28 ( G ), and NEK9 and CTTN ( H ) in AGS and MKN45 cells. I Representative images of IF staining for NEK9 (red) and CTTN (green), showing the colocalization in AGS and MKN45 cell, Scale, 20 μm. J and K The function of NEK9, TRIM28, TRIM28 mutation, CTTN and CTTN mutation on cell motility were evaluated via in vitro Transwell assay. L The phosphorylation levels of TRIM28 and CTTN were assessed with SLIT2 treatment (100 ng/ ml, 24 hours) in AGS and MKN45 cells via Western blot analysis. TRIM28 mut: mutant TRIM28 (S473 and S824), CTTN mut: mutant CTTN (S405, S417, S418 and S447), *** P < 0.001. TRIM28 tripartite motif containing 28, CTTN cortactin.

Article Snippet: CAF cancer-associated fibroblasts, GC gastric cancer, ROBO1, roundabout guidance receptor 1, SLIT2Δ, SLIT2 knockdown in CAFs.

Techniques: Transfection, Over Expression, Mutagenesis, Plasmid Preparation, Migration, In Vitro, Transwell Assay, Staining, Phospho-proteomics, Western Blot, Kinase Assay, Co-Immunoprecipitation Assay

Journal: Cell Death & Disease

Article Title: Cancer associated fibroblast derived SLIT2 drives gastric cancer cell metastasis by activating NEK9

doi: 10.1038/s41419-023-05965-z

Figure Lengend Snippet: NEK9 kinase domain and the intracellular domain of ROBO1 was enhanced by cancer-associated fibroblast-derived SLIT2. NEK9 directly phosphorylated TRIM28 (S473) and CTTN (S417). Phosphorylation on S473 of TRIM28 increased its transcriptional activities on STAT3, p100 and CTTN. TRIM28, STAT3 and p100 functioned synergistically to transcriptionally activate CTTN. Increased expression and phosphorylation levels of CTTN led to cytoskeletal reorganization and gastric cancer metastasis. ROBO1, roundabout guidance receptor 1; TRIM28, tripartite motif containing 28; CTTN, cortactin.

Article Snippet: CAF cancer-associated fibroblasts, GC gastric cancer, ROBO1, roundabout guidance receptor 1, SLIT2Δ, SLIT2 knockdown in CAFs.

Techniques: Derivative Assay, Phospho-proteomics, Expressing