Materials Quercetin

was purchased from Cayman Chemicals (

Materials Quercetin

was purchased from Cayman Chemicals (Ann Arbor, MI), with all other chemicals and reagents being purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO). Gene expression reagents were obtained from Bio-Rad (Hercules, CA). Primers were designed and purchased along with TRIzol® from Life Technologies (Carlsbad, CA). Methods Initially animals were acclimatized to the housing facility and the use of the treadmill instrument prior to starting the actual protocol. After 30 days of treatment the animals were fasted overnight (>12 hours), sacrificed with 100% CO2 exposure, and blood was collected via cardiac puncture. The plasma was collected after centrifugation at 4°C at 3000 rpm for 20 min and frozen at −80°C until assayed. The aorta and liver were perfused with cold phosphate buffered saline selleck inhibitor (PBS) prior to being harvested. All tissues were instantaneously frozen in liquid nitrogen following collection and stored at −80°C until assayed. Assessment of atherosclerotic lesions At the completion of the livers perfusion and tissue collection the aorta was kept wet with cold PBS through the dissection process which was performed under a stereomicroscope from the iliac bifurcation up to the heart, including the beginning of the brachiocephalic, carotid, and subclavian arteries. Pictures of the aorta were obtained using

a digital camera. Lesion area size was quantified TSA HDAC using Image J software [31]. The ADP ribosylation factor lesion area was marked on the pictures under direct microscopic observation and quantified. Quantitative real-time PCR (qPCR) Liver RNA was

extracted using TRIzol according to the manufacturer’s protocol and the quantity was measured by Qubit (Life Technologies, Carlsbad, CA). cDNA was generated from 10–100 ng of total RNA and 1/20th of the sample was taken for qPCR. cDNA synthesis and qPCRs were performed with SYBR GreenER Two-Step qRT-PCR Kit according to the manufacturer’s protocol. qPCR was run in 20 μL of reaction mixture in sealed 96-well plates with iScriptTM Reverse Transcription Supermix and SsoFastTM EvaGreen® Supermix on an RTPCR MyiQTM2 system (Bio-Rad; Hercules, CA). Threshold cycle (CT) was determined by Bio-Rad iQ5 v.2.1 software. The melting curve and efficiency were assessed for all primer pairs. The level of mRNA was calculated using glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as an internal control gene. Data are expressed as fold induction of mRNA level in one group compared to another. Enzyme-Linked Immunosorbent Assay (ELISA) Plasma TNF-α, monocyte chemoattractant protein (MCP)-1, and interleukin (IL)-17α levels were determined according to manufacturer protocols by ELISA kits purchased from BioLegend (San Diego, CA). Statistical analysis All data are Emricasan purchase presented as mean ± SD. Statistical significance for differences in lesion areas were evaluated using Student’s t-test.

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