Journal: Cells

Article Title: EF24, a Curcumin Analog, Reverses Interleukin-18-Induced miR-30a or miR-342-Dependent TRAF3IP2 Expression, RECK Suppression, and the Proinflammatory Phenotype of Human Aortic Smooth Muscle Cells

doi: 10.3390/cells13201673

Figure Lengend Snippet: Targeting TRAF3IP2 or RECK overexpression blunts IL-18-induced ASMC proliferation and migration. ( A – E ) Silencing TRAF3IP2 inhibits IL-18-induced ASMC proliferation ( B ) and migration ( C ). ASMC were transduced with adenovirus-expressing shRNA targeting human TRAF3IP2 (moi 10 for 48 h), made quiescent, and then exposed to IL-18 (10 ng/mL; experimental design in ( A )). ASMC proliferation was assessed after 48 h of IL-18 addition using the CyQUANT Cell proliferation assay ( B ), and migration after 18 h using Boyden chamber assay ( C ). ASMCs migrating to the lower surface of the membrane were counted in 10 different fields and summarized as mean ± SEM. ( B , C ) * p < at least 0.01 vs. Untreated; † p < 0.01 vs. IL-18 or IL-18+GFP (n = 6). Knockdown of TRAF3IP2 was confirmed by RT-qPCR using a TaqMan™ probe ( D ) and Western blotting ( E ). ( D , E ) * p < 0.01 vs. untreated (n = 3). ( F , G ) Dose-dependent effects of Ad.RECK on RECK expression (experimental design in ( F )). Induction of RECK following adenoviral transduction was confirmed by Western blotting with tubulin serving as an internal control ( G ). ( H – K ) Forced expression of RECK inhibits IL-18-stimulated ASMC proliferation and migration. ASMCs were transduced with adenovirus-expressing human RECK cDNA (moi 10 for 24 h), made quiescent, and then treated with IL-18 (experimental design in ( H )) and analyzed for proliferation ( I ) and migration ( J ) as in ( B , C ). ( C , J ) The insets show representative images of Matrigel™ Transwell invasion. Scale bar: 20 μM. ( E , G , K ) While a representative immunoblot is shown, the intensities of immunoreactive bands from three ( E ), four ( G ) and three ( K ) independent experiments were semiquantified by densitometry and are summarized on the right. ( I ) * p < at least 0.01 vs. Untreated; † p < 0.01 vs. IL-18 or IL-18+GFP (n = 6); ( J ) * p < at least 0.01 vs. Untreated; † p < 0.01 vs. IL-18 or IL-18+eGFP (n = 4); ( K ) * p < 0.01 vs. Untreated; † p < 0.05 vs. IL-18 or IL-18+eGFP (n = 3).

Article Snippet: Adenoviral vector-expressing human RECK cDNA (GenBank accession # NM_021111) was also custom-generated at Vector Biolabs.

Techniques: Over Expression, Migration, Transduction, Expressing, shRNA, CyQUANT Assay, Proliferation Assay, Boyden Chamber Assay, Membrane, Knockdown, Quantitative RT-PCR, Western Blot, Control

Journal: Cells

Article Title: EF24, a Curcumin Analog, Reverses Interleukin-18-Induced miR-30a or miR-342-Dependent TRAF3IP2 Expression, RECK Suppression, and the Proinflammatory Phenotype of Human Aortic Smooth Muscle Cells

doi: 10.3390/cells13201673

Figure Lengend Snippet: Ectopic expression of RECK blunts IL-18-induced inhibition in the expression of SMC markers and the induction of proinflammatory phenotype markers. ( A – F ) Forced expression of RECK restores IL-18-induced suppression of SMC markers and inhibits the induction of proinflammatory phenotype markers. ASMCs were transduced with adenovirus-expressing human RECK cDNA (moi10 for 24 h), made quiescent, and then treated with IL-18 at 10 ng/mL for 48 h (experimental design in ( A )). The expression levels of SMC markers ACTA2 ( B , C ) and MYH11 ( D , E ) were analyzed by RT-qPCR ( B , D ) and Western blotting ( C , E ). The proinflammatory phenotype markers Galectin 3, Olr1, VCAM, CCL2, IL-6, IL-8, and TNF-α were analyzed by RT-qPCR using TaqMan™ probes ( F ). ( C , E ) While a representative immunoblot is shown, the intensities of immunoreactive bands from three independent experiments were semiquantified by densitometry and are summarized on the right. ( B , D , F ) * p < at least 0.01 vs. Untreated; † p < 0.01 vs. IL-18 or IL-18+eGFP (n = 5). ( C , E ) * p < 0.05 vs. Untreated; † p < 0.05 vs. IL-18 or IL-18+eGFP (n = 3).

Article Snippet: Adenoviral vector-expressing human RECK cDNA (GenBank accession # NM_021111) was also custom-generated at Vector Biolabs.

Techniques: Expressing, Inhibition, Transduction, Quantitative RT-PCR, Western Blot