Ribozol

Microglia cell line (SIM-A9) was used and cultured as previously reported [33 (link)]. Briefly, microglial cells (800, 000 cells per well) were cultured in 6-well plates (Sarstedt, Inc., Newton, NC, USA) with DMEM/F12 (1:1) supplemented with 10% fetal bovine serum (FBS), 5% of horse serum (HS), and 1% penicillin/streptomycin. After 24 h, the cells were starved with DMEM/F12 (1:1) free of FBS and HS for 6 h. Then, microglial cells cultures in presence or absence of 100 μM of (6R)-5,6,7,8-Tetrahydrobiopterin dihydrochloride (BH4; Sigma, Cat. T4425) were exposed to hyperoxia (75% oxygen and 25% nitrogen; Hyp-MGCM) in a modular incubator chamber (Billups-Rothenberg, Inc) and maintained in a humidified CO₂ incubator at 37 °C for 24 h. Microglial cells in matching controls (Nor-MGCM) were incubated at 37 °C in an incubator with 95% air and 5% CO₂ and collected at the same time point. Cell lysates were quickly processed for RNA using RiboZol (Amresco, N580, OH, USA). The conditioned media was stored at −80 and later used in choroidal explant assay.

HepG2 cells were seeded in 6-well plates at a density of 4×10⁵/ml. Twenty-four hours after plating, cells were incubated with either 0.1% DMSO or 0.5 μg/ml doxorubicin (DOX) in Dulbecco's Modified Eagle Medium supplemented with 10% FBS. Total RNA was extracted using RiboZol (Amresco, Solon, OH, USA) for which integrity was verified by agarose gel electrophoresis. Afterwards, cDNA was prepared from 1 μg of total RNA using the SuperScript II Reverse transcriptase (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. qPCR was performed from 1:10 dilution of the converted cDNA mixed with PerfeCTa SYBR Green SuperMix, UNG, Low ROX (Quanta Biosciences, Gaithersburg, MD, USA) using the MX3000p real-time thermal cycler (Agilent, Mississauga, On, Canada). For each gene of interest, dissociation curves and agarose gel electrophoresis were performed to ensure unique PCR product. Arbitrary unit was determined from PCR duplicates for each sample using the TATA box binding protein (TBP) as a normalizer. Oligonucleotides sequences used are listed in Table 2. Error bars are representative of four independent experiments analyzed in duplicate.

RNA was collected from cells using a Ribozol (Amresco) extraction method. RNA was treated with DNaseI (Sigma-Aldrich) per supplier protocol. cDNA was synthesized using qScript cDNA SuperMix (Quantigene). Amplification of cDNA was measured using Lightcycler 480 SYBR Green I Master (Roche) with the CT method and normalized to L32. Primers used were L32F 5-AAGCGAAACTGGCGGAAAC-3, L32R 5-TAACCGATGTTGGGCATCAG-3, CIITAF 5-ACACCTGGACCTGGACTCAC-3, CIITAR 5-GCTCTTGGCTCCTTTGTCAC-3, TGF-BF 5-CTGCTGAGGCTCAAGTTAAAAGTG-3, TGF-BR 5-CAGCCGGTTGCTGAGGTA-3. JHMV-F 5-CGAGCCGTAGCATGTTTATCTA-3, JHMVR 5-CGCATACACGCAATTGAACATA-3. TLR4F 5-TGGGTCAAGGAACAGAAGCAGT-3 TLR4R 5-AATCCAACACTAAGGAGGTATTCA-3.

RAW Blue cells were purchased from InvivoGen (San Diego, CA), used at passages under 15, and cultured in DMEM growth medium supplemented with 10% serum, 50 U/mL penicillin and 200 mg/mL zeocin. Cells were grown in regular conditions (37°C, 5% CO₂), serum starved overnight, and treated with 100 ng/mL IL-1β for 4 h. Cells were, respectively pre-incubated for 30 min with peptides 1 or 2 (10⁻⁶ M) or Kineret (1.0 mg/mL) to reach equilibrium prior to the experiments (n = 4 each treatment). Cells were harvested and incubated for 5 min in RIBOzol (AMRESCO). RNA was extracted according to manufacturer's protocol and RNA concentration and integrity were measured with a NanoDrop 1,000 spectrophotometer. A total of 500 ng of RNA was used to synthesize cDNA using iScript Reverse Transcription SuperMix (Bio-Rad, Hercules, CA). Primers (Table 1) were designed using National Center for Biotechnology Information Primer Blast. Quantitative gene expression analysis was performed using the Stratagene MXPro3000 (Stratagene) with SYBR Green Master Mix (Bio-Rad). Gene expression levels were normalized to 18S universal primer (Ambion Life Technology, Burlington ON, Canada). Genes analyzed include IL1β and PTGHS2 [Prostaglandin H synthetase 2 or cyclooxygenase-2 (COX-2)]. Data are representative of 3 experiments (each with n = 4 per treatment group).

Leaf tissue was collected from 4-week old Arabidopsis plants that were syringe-infiltrated with 10 µM flg22 (Genscript) or 10 µM elf18 (Genscript).
Pathogen treatments were performed using 4-week old plants that were syringe-infiltration with Psm ES4326/avrRpm1 (OD_600nm = 0.1), spray inoculated with Pst DC3000 (OD_600nm = 0.2, 0.02% Silwet L-77) or Pst DC3118 (OD_600nm = 0.2, 0.02% Silwet L-77). Untreated root, shoot leaf, shoot, and flower tissues were collected to determine the basal levels of tarnscripts. We extracted total RNA from the collected samples using RiboZol (AMRESCO). Possible genomic DNA contamination was eliminated using DNase I (Ambion). Formaldehyde agarose gel preparation, quantification, electrophoresis and samples prepration/loading were done following the RNeasy Plant Mini® Kit QIAGEN protocol. SuperScript III first-strand RT-PCR kit (Invitrogen) was used to convert mRNA into cDNA. We used GoTaq qPCR Master Mix (Promega) to perform qRT-PCR using gene-specific primers in a RealPlex S MasterCycler (Eppendorf). Primers used in this study are listed in the Supplementary Table 1.

RNA expression of GDF15 was analyzed in a total of 25 samples based on RNA quality; normal (n = 5), pre-obese (n = 5), obese class I (n = 5), class II (n = 5), and class III (n = 5). RNA was isolated from atrial tissue (~30 mg) using Ribozol (Amresco, OH, USA), followed by chloroform extraction (13 (link)). RNA integrity was assessed in all cases (Synergy H4, Biotek, VT, USA), and 1 μg of RNA was used to synthesize cDNA (qScript cDNA supermix, Quanta Biosciences). For quantitative Polymerase Chain Reaction (qPCR), 2 μl of cDNA was mixed with SYBR Green PCR supermix (AB1323A, Thermo Fisher Scientific) and primers (Human GDF15 Forward 5′-CTCCAGATTCCGAGAGTTGC-3′ and Reverse 5′-AGAGATACGCAGGTGCAGGT-3′) and the reaction was performed in duplicate with 40 cycles (13 (link)). Human PPIA (Forward 5′-ATGTGTCAGGGTGGTGACTTC-3′ and Reverse 5′-GCCATCCAACCACTCAGTCTT-3′) was used as a reference gene and results expressed as a ratio of Cycle threshold (Ct) values of the target and reference genes.