Cox 2

RNA was isolated employing RNeasy Mini Kit (Cat. N° 74104, Qiagen) and treated with RNAse-Free DNase Set (Cat. N° 79254, Qiagen). One-microgram RNA was transcribed into cDNA using iScript^TM CDNA Synthesis Kit (Cat. N° 170-8891, BioRad) according to the manufacturer protocol.
TaqMan real-time polymerase assays were performed utilizing TaqMan Universal PCR Master Mix (4304437, Applied Biosystem) and the following TaqMan Gene Expression assays: Bcl-2 (Mm00477631_m1, Applied Biosystems); Caspase-3 (Mm01195085_m1, Applied Biosystems); Leptin Receptor (Mm00440181_m1, Applied Biosystems); Caspase-9 (Mm00516563_m1, Applied Biosystems); Rank-L (Mm00441906_m1, Applied Biosystems); Runx-2 (Mm00501584_m, Applied Biosystems); Caspase-8 (Mn00802247_m1, Applied Biosystems); Cox-2 (Mm03294838_g1); Pges (Mm00452105_m1); Sost (Mm00470479_m1). Target gene expressions were normalized to the expression of β-Actin (Mm00607939_s1, Applied Biosystems) as housekeeping gene. Thresholds were amplified and detected using CFX96TM Real-Time System Cycler (Bio-Rad). Results were analyzed using the Bio-Rad CFX Manager 3.1 software. Each experiment was repeated at least three times.

Total RNA was extracted from 100 mg of arterial and cardiac tissues using the Chomczynski method [52 (link)] and quantified with a Nanodrop2000 spectrometer (Thermo Fisher Scientific, Hampton, NH, USA). Afterward, 1µg of total RNA was retrotranscripted into cDNA using a high-capacity cDNA reverse transcription kit (Applied Biosystems, Foster City, CA, USA).
Fluorogenic (FAM, VIC) primers designed by the Assay-onDemandTM mouse gene expression products were used (Applied Biosystems; COX2: Rn00585003_m1; glutathione reductase (GRS): Rn01482159_m1; glutathione peroxidase (GPX3): Rn00574703_m1; iNOS: Rn06325074_m1; tumor necrosis factor Alpha (TNFα): Rn00562055_m1; superoxide dismutase (SOD1): Rn00566938_m1; Interleukin-6 (IL-6): Rn01410330_m1; Interleukin-10 (IL-10): Rn01483987_m1; NADPH oxidase 1 (NOX1): Rn00586652_m1; NOX4: Rn00585380_m1; and 18S: Rn03928990_g1).

Quantitative RT-PCR was performed as described previously [21] (link). Intron overlapping primer sets and minor groove binder probes were purchased as Assay-on-Demand from Applied Biosystems (Nieuwekerk a/d IJssel, The Netherlands): housekeeping gene GAPDH (assay ID Hs99999905_m1), COX-2 (assay ID Hs00153133_m1), VEGF (Hs00173626_m1), VEGFR-1 (Hs00176573_m1), VEGFR-2 (Hs00176676_m1), Tie2 (assay ID Hs00176096_ml), Ang-1 (assay ID Hs00181613_ml), Ang-2 (assay ID Hs00169867_ml).

NF-κB and COX-2 expression levels were measured using the immunofluorescent method. NF-κB (ThermoFisher Scientific, PA5-16545, Waltham, MA, USA), COX-2 (Invitrogen, PA1-9032, Carlsbad, CA, USA), FITC-conjugated IgG (Jackson Immunoresearch, 315-095-003, West Grove, PA, USA), Alexa Fluor 555-conjugated IgG (ThermoFisher Scientific, A-21127), and DAPI (ThermoFisher Scientific, 62249) were used. A K1-Fluo Confocal Microscope (Nanoscope System, Daejeon, Korea) was used for image acquisition and for analyzing the fluorescent intensity.

Total RNA was isolated from the spinal cords using a Nucleospin RNA II kit^® (Macherey-Nagel, Düren, Germany). First-strand cDNA synthesis and real-time reverse transcription-polymerase chain reaction (RT-PCR) were conducted with One Step CYBR™ RT-PCR Kit II (Takara Bio, Shiga, Japan) according to the manufacturer’s instructions. RT-PCR assays were conducted using the 7300 Real-Time PCR System (Applied Biosystems, CA, USA). The PCR primers of 18S and IL-6 for mice were obtained from Qiagen (Valencia, CA, USA) (Catalog Numbers: 18S mouse; QT02448075, IL-6 mouse; QT00098875). IL-1β, TNFα, COX-2, and IL-36γ were obtained from Invitrogen (CA, USA). Primer sequences were as follows: IL-1β 5′-ATGAGGACATGAGCACCTTC-3′ (forward) and 5′-CATTGAGTTGGAGAGCTTTC-3′ (reverse), TNFα 5′-TCGTAGCAAACCACCAAGTG-3′ (forward) and 5′-CCTTGAAGAGAACCTGGGAGT-3′ (reverse), COX-2 5’ -TGAGCAAC- TATTCCAAACCAGC-3’ (forward) and 5’ -GCACGTAGTCTTCGATCAC- TATC-3’ (reverse), and IL-36γ 5’ -AGAGTAACCCCAGTCAGCGTG-3’ (forward) and 5’ -AGGGTGGTGGTACAAATCCAA-3’ (reverse). For each target mRNA, the fold changes in expression were calculated relative to 18S rRNA.

RT-qPCR was run on a LightCycler 480 instrument (Roche Diagnostics, Mannheim, Germany) using Maxima SYBR Green PCR Master Mix (Thermo Fisher Scientific) as described [22 (link)]. Primers (Il6, Il1β, Tnfα, iNos, Cox2, peptidylprolyl isomerase B (Ppib)) were purchased from Invitrogen (Karlsruhe, Germany; Supplementary Table S1). Results were analyzed using LightCycler software version 1.5.0.39 (Roche Applied Science, Mannheim, Germany). The fold change of mRNA expression was normalized to the expression of the reference gene Ppib. Quantitative analysis was performed using the 2^-ΔΔCT method.