Journal: International Journal of Cancer

Article Title: Integrin β6 expression in colorectal cancer cells promotes liver metastasis through enhanced adhesion to endothelial fibronectin

doi: 10.1002/ijc.35504

Figure Lengend Snippet: Integrin β6 is a proteolysis‐resistant mediator of CRC tumour cell adhesion to endothelial cells. (A) Lysates of HT‐29 and DLD‐1 cells harvested in the presence of 0.05% trypsin or 5 mM EDTA‐PBS were collected, and the expression of integrins αv, β6, β1 and β5 was analysed using western blotting. Blots show the full‐length proteins as well as the trypsin‐induced protein fragments. GAPDH was used as a loading control. (B) Adhesion of cytopainter‐stained ITGB6 KO or NT cells harvested by trypsinization or 5 mM PBS‐EDTA to an IL‐1β‐stimulated HUVEC monolayer under static conditions. Arrow points towards an adherent cell. Scale bar: 250 μm. The quantification is shown below. Mann–Whitney U test; ns, not significant; ** p < 0.01. (C) The cancer‐related survival (60‐month cut‐off) of CRC patients who did not receive neoadjuvant therapy and had no residual tumour (R0) was explored. Based on MMP11 and MMP12 RNA expression levels, patients were divided into high‐ and low‐MMP groups. ITGB6 ‐dependent survival was investigated in these groups. The number of patients per group is indicated by ‘ n ’. Cox proportional hazards survival regression. (D) Recombinant integrins αvβ6, αvβ1, and αvβ5 were incubated with MMP‐12 at an enzyme‐to‐substrate ratio of 1:10 for 1 h. Proteolytic cleavage of integrins was evaluated via western blotting.

Article Snippet: MMP‐12 (R&D Systems, Catalogue #: 917‐MPB) was activated in a concentration of 50 ng/μL by incubation in TCN buffer (10 mM Tris, 150 mM NaCl, 5 mM CaCl2, pH 7.5) at 37°C for 2 h. Following enzyme activation, the recombinant human integrins (integrin αvβ6; Catalogue #: T6‐H52E1, integrin αvβ1; Catalogue #: IT1‐H52E1, integrin αvβ5; Catalogue #: IT5‐H52W5, AcroBiosystems, Newark, DE, USA) were added in an enzyme‐to‐substrate ratio of 1:10 in TCN buffer for 1 h (MMP‐12) at 37°C.

Techniques: Expressing, Western Blot, Control, Staining, MANN-WHITNEY, RNA Expression, Recombinant, Incubation

Journal: BMB Reports

Article Title: SPINK1 promotes cell growth and metastasis of lung adenocarcinoma and acts as a novel prognostic biomarker

doi: 10.5483/bmbrep.2018.51.12.205

Figure Lengend Snippet: Fig. 3. SPINK1 enhances the migration and invasion of LAC cells by up-regulating MMP12. (A) MMPs expressions were analyzed by qRT-PCR in A549 cells and H1975 cells transfected with scramble or SPINK1 siRNAs. (B) MMP12 protein expressions were analyzed by Western blot in A549 cells and H1975 cells transfected with scramble or SPINK1 siRNA 1. (C) MMP12 protein levels were measured by Western blot in PC9 cells and H1299 cells transfected with control or SPINK1. (D) MMP12 mRNA expression in xenograft tissues was measured by qPCR. (E) The correlation between SPINK1 expression and MMP12 expression in LAC tissues was analyzed with Pearson correlation. (F) The interference effect of MMP12 siRNA was verified by Western blot in PC9 and H1299 cells stably overexpressing SPINK1. The number of migrating and invasive cells was counted (G). (H) Recombinant human MMP12 (rhMMP12) or control IgG (cIgG) at a concentration of 50 ng/ml was added back into SPINK1 knockdown A549 and H1299 cells, and the number of migrating and invasive cells was counted. *P ＜ 0.05, **P ＜ 0.01, ***P ＜ 0.001.

Article Snippet: Recombinant human MMP12 (rhMMP12, 917-MPB) and the control IgG (cIgG, 1-001-A) were purchased from R&D Systems.

Techniques: Migration, Quantitative RT-PCR, Transfection, Western Blot, Control, Expressing, Stable Transfection, Recombinant, Concentration Assay, Knockdown