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: TRAF3IP2 knockdown restores SMC marker expression and inhibits ASMC proinflammatory phenotype without affecting cell viability. ( A – G ) Silencing TRAF3IP2 restores IL-18-mediated suppression in SMC markers, but inhibits the expression of proinflammatory phenotype markers, without significantly modulating cell viability. ASMCs were transduced with adenoviral TRAF3IP2 shRNA (moi10 for 48 h), made quiescent and then treated with IL-18 (10 ng/mL for 48 h; experimental design in ( A )). Expressions of the SMC markers ACTA2 ( B , C ) and MYH11 ( D , E ) were analyzed by both 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 ). Cell viability was assessed by analyzing cleaved caspase-3 levels using a commercially available Caspase-3 (Cleaved) Human ELISA ( G ). H 2 O 2 (100 μM for 18 h) served as a positive control and induced a significant increase in cleaved capase-3 levels. ( 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 , G ) * p < at least 0.01 vs. Untreated; † p < 0.01 vs. IL-18 or IL-18+GFP (n = 6 or 7). ( C , E ) * p < 0.05 vs. Untreated; † p < 0.05 vs. IL-18 or IL-18+GFP (n = 3).

Article Snippet: Two micrograms of total RNA with an RNA integrity number of 9.0 or greater (scale = 1–10) were reverse-transcribed into single-stranded cDNA and used in RT-qPCR employing Applied Biosystems™ TaqMan ® probes— TRAF3IP2 (Assay ID: Hs00974570_m1), RECK (Hs01019185), ACTA2 (Hs00426825_g1), MYH11 (Hs00975796_m1), Galectin 3 ( LGALS3 ; Hs03680062_m1), VCAM1 (Hs00164932_m1), OLR1 (Hs01552593_m1), CCL2 (MCP1; Hs07292220_s1), Il6 (Hs00174131_m1), Il8 (CXCL8; Hs00174103_m1), and TNF-α (Hs00174128_m1).

Techniques: Knockdown, Marker, Expressing, Transduction, shRNA, Quantitative RT-PCR, Western Blot, Enzyme-linked Immunosorbent Assay, Positive 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: Two micrograms of total RNA with an RNA integrity number of 9.0 or greater (scale = 1–10) were reverse-transcribed into single-stranded cDNA and used in RT-qPCR employing Applied Biosystems™ TaqMan ® probes— TRAF3IP2 (Assay ID: Hs00974570_m1), RECK (Hs01019185), ACTA2 (Hs00426825_g1), MYH11 (Hs00975796_m1), Galectin 3 ( LGALS3 ; Hs03680062_m1), VCAM1 (Hs00164932_m1), OLR1 (Hs01552593_m1), CCL2 (MCP1; Hs07292220_s1), Il6 (Hs00174131_m1), Il8 (CXCL8; Hs00174103_m1), and TNF-α (Hs00174128_m1).

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

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: EF24 reverses ASMC proinflammatory phenotype without affecting cell viability. ( A – F ) Pretreatment with EF24 restores the IL-18-induced suppression of SMC markers and inhibits the expression of proinflammatory phenotype markers, without significantly modulating cell viability. Quiescent ASMCs were treated with EF24 (2.5 μM for 1 h in DMSO) prior to the addition of IL-18 at 10 ng/mL for 48 h (experimental design in ( A )). The expression levels of SMC markers ACTA2 (( B ) mRNA, ( C ) protein levels) and MYH11 (( D ) mRNA, ( E ) protein levels) were analyzed by RT-qPCR and Western blotting, and those of proinflammatory phenotype markers Galectin 3, Olr1, VCAM, CCL2, IL-6, IL-8, and TNF-α ( F ) were analyzed by RT-qPCR using TaqMan™ probes. Cell viability was assessed by analyzing cleaved caspase-3 levels using a Caspase-3 (Cleaved) Human ELISA kit ( G ). H 2 O 2 (100 μM) for 24 h served as a positive control and induced a significant increase in cleaved capase-3 levels. ( 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 , G ) * p < at least 0.01 vs. Untreated; † p < at least 0.05 vs. IL-18 or IL-18+DMSO (n = 5–6), ( C – E ) * p < 0.05 vs. Untreated; † p < 0.05 vs. IL-18 or IL-18+DMSO (n = 3).

Article Snippet: Two micrograms of total RNA with an RNA integrity number of 9.0 or greater (scale = 1–10) were reverse-transcribed into single-stranded cDNA and used in RT-qPCR employing Applied Biosystems™ TaqMan ® probes— TRAF3IP2 (Assay ID: Hs00974570_m1), RECK (Hs01019185), ACTA2 (Hs00426825_g1), MYH11 (Hs00975796_m1), Galectin 3 ( LGALS3 ; Hs03680062_m1), VCAM1 (Hs00164932_m1), OLR1 (Hs01552593_m1), CCL2 (MCP1; Hs07292220_s1), Il6 (Hs00174131_m1), Il8 (CXCL8; Hs00174103_m1), and TNF-α (Hs00174128_m1).

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Enzyme-linked Immunosorbent Assay, Positive Control