recombinant mmp12 r d systems  (R&D Systems)

Journal: Cell Death & Disease

Article Title: RON receptor tyrosine kinase as a critical determinant in promoting tumorigenic behaviors of bladder cancer cells through regulating MMP12 and HIF-2α pathways

doi: 10.1038/s41419-024-07245-w

Figure Lengend Snippet: A Significant alterations in mRNA expression were observed in 5637-shRON cells compared to 5637-NC cells, as determined by RNA-seq analysis. B Through KEGG and GO analysis, as well as combining annotations in the GENE database, 13 candidate genes closely associated with migration and invasion were identified among genes positively correlated with RON expression. C Validation of candidate genes positively correlated with RON expression in 5637-shRON, 5637-NC, J82-oeRON, and J82-NC groups was conducted using qRT-PCR. D The protein expression levels of MMP12 were assessed in 5637-shRON, 5637-shNC, J82-oeRON, and J82-oeNC cells by western blot. Experiments in C , D were repeated three times ( n = 3).

Article Snippet: Mammalian expression vectors containing the full-length human RON and MMP12 cDNA were purchased from Genechem (Shanghai, China).

Techniques: Expressing, RNA Sequencing, Migration, Biomarker Discovery, Quantitative RT-PCR, Western Blot

Journal: Cell Death & Disease

Article Title: RON receptor tyrosine kinase as a critical determinant in promoting tumorigenic behaviors of bladder cancer cells through regulating MMP12 and HIF-2α pathways

doi: 10.1038/s41419-024-07245-w

Figure Lengend Snippet: A J82 cells were transduced with siRNA targeting MMP12 and an overexpression plasmid for RON. Western blot analysis was utilized to confirm the expression of MMP12. B The migratory capacity was assessed through a wound-healing assay. C The invasive potential was evaluated using a trans-well assay. D In total, 5637 cells were transduced with an overexpression plasmid for MMP12 and a shRON plasmid. Western blot analysis was employed to validate the expression of MMP12. E The migratory ability was assessed through a wound-healing assay. F The invasion ability was evaluated by trans-well assay. The scale bar above is all 1 µm. Actin-Tracker Red-555 and DAPI were utilized for staining the cytoskeleton and nucleus, respectively, with representative images displayed in Fig. ( G ) and ( H ). The scale bar measures 500 µm. I , J Western blot analysis was used to confirm the expression of N-cadherin and vimentin. All experiments above were repeated three times ( n = 3).

Article Snippet: Mammalian expression vectors containing the full-length human RON and MMP12 cDNA were purchased from Genechem (Shanghai, China).

Techniques: Transduction, Over Expression, Plasmid Preparation, Western Blot, Expressing, Wound Healing Assay, Staining

Journal: Cell Death & Disease

Article Title: RON receptor tyrosine kinase as a critical determinant in promoting tumorigenic behaviors of bladder cancer cells through regulating MMP12 and HIF-2α pathways

doi: 10.1038/s41419-024-07245-w

Figure Lengend Snippet: A Co-immunoprecipitation was employed to demonstrate the lack of direct interaction between RON and MMP12. B – D The mRNA expression levels of HIF-2α in 5637-shRON, 5637-NC, J82-oeRON, and J82-NC cells were quantified using quantitative real-time polymerase chain reaction (qRT-PCR), and confirmed by immunofluorescence staining and western blot analysis, respectively. E Transfection of 5637-shRON cells with a HIF-2α plasmid and J82-oeRON cells with HIF-2α-targeting siRNA was performed, followed by western blot analysis to assess the expression levels of HIF-2α and MMP12. F J82-oeRON cells were subjected to a 24-h period of starvation, followed by washing with ice-cold PBS to remove basal phosphorylation. Subsequently, the cells were stimulated with the RON-specific ligand MSP for 30 min, and western blot analysis was performed to assess the phosphorylation levels of ERK and JNK. G , H J82-oeRON cells were treated with varying concentrations of ERK inhibitors or JNK inhibitors for 24 h, after which the cells were harvested to evaluate the expression of HIF-2α and MMP12 by western blot. I J82-oeRON cells were subjected to treatment with or without 20 µM JNK inhibitors for a duration of 24 h, followed by the addition of 200 nM bortezomib 8 h prior to cell collection. Subsequently, the cells underwent immunoprecipitation using an anti-HIF-2α antibody, and the ubiquitination level of HIF-2α was assessed through immunoblotting with an anti-ubiquitin antibody. All experiments above were repeated three times ( n = 3).

Article Snippet: Mammalian expression vectors containing the full-length human RON and MMP12 cDNA were purchased from Genechem (Shanghai, China).

Techniques: Immunoprecipitation, Expressing, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Immunofluorescence, Staining, Western Blot, Transfection, Plasmid Preparation, Phospho-proteomics, Ubiquitin Proteomics

Journal: Cell Death & Disease

Article Title: RON receptor tyrosine kinase as a critical determinant in promoting tumorigenic behaviors of bladder cancer cells through regulating MMP12 and HIF-2α pathways

doi: 10.1038/s41419-024-07245-w

Figure Lengend Snippet: The inhibition of miR-659-3p resulted in the prevention of RON 3’UTR binding and subsequent RON mRNA degradation in the context of cancer progression. The overexpression of RON can lead to its activation through spontaneous phosphorylation, initiating downstream activation of the JNK signaling pathway, which in turn inhibits HIF-2α ubiquitination and stabilizes its expression. HIF-2α serves as a transcription factor for MMP12, promoting its expression and ultimately enhancing the migration and invasion of bladder cancer cells.

Article Snippet: Mammalian expression vectors containing the full-length human RON and MMP12 cDNA were purchased from Genechem (Shanghai, China).

Techniques: Inhibition, Binding Assay, Over Expression, Activation Assay, Phospho-proteomics, Ubiquitin Proteomics, Expressing, Migration