Stepone machine

Total RNA from 1 × 10⁶ trophozoites incubated or not with ACh 1, 0.01, 0.0001, or 0.000001 µM for 1 h was isolated with the Direct-zol RNA Miniprep kit (Zymo Research, Irvine, California, USA), following the manufacturer’s protocol. Reverse transcription was performed with 500 ng of total RNA from E. histolytica using the Revert Aid First Strand cDNA Synthesis Kit (Thermo Fisher Scientific), and gene expression was measured using 50 ng of cDNA by quantitative real-time PCR with the Maxima SYBR Green qPCR Master Mix (2×) (Thermo Scientific, California, USA) in a Step One machine (Applied Biosystems, Thermo Fisher Scientific, California, USA) using the following program: 50°C for 2 min, 95°C for 3 min, and 40 cycles of 95°C for 30 sec and 56°C for 30 sec. Oligonucleotides were designed to target genes encoding E. histolytica cysteine proteinase 2 (ehcp-a2), cysteine proteinase 5 (ehcp-a5), amebapore C, and Gal/GalNAc lectin heavy subunit (Table 1). Expression levels were normalized to that of the housekeeping gene α-tubulin and differences were determined by employing the 2^-ΔΔCt method (Livak and Schmittgen, 2001 (link)), using the StepOne machine.

RNA was isolated using an RNeasy kit with on-column DNase treatment (Qiagen, Germantown, MD, USA). cDNA was generated using the Multiscribe Reverse Transcriptase kit (Thermo Fisher, Waltham, MA, USA). qPCR analysis was performed with Fast SYBR Green reagent (Invitrogen, Waltham, MA, USA) using a StepOne machine (Applied Biosystems, Waltham, MA, USA). Primers were validated by performing a standard melt curve analysis, and the results are listed in the Key Resources Table.

Total RNA was isolated from 3T3-L1 cells using an ISOGEN (Nippon Gene, Tokyo, Japan), with removal of contaminating DNA by a TURBO DNA-Free Kit (Invitrogen), and reverse transcription (RT) was performed by ReverTra Ace (Toyobo, Osaka, Japan). The resulting cDNA was subjected to real-time PCR with a StepOne machine and Power SYBR Green PCR master mix (Applied Biosystems, Foster City, CA). The primers used for the amplification are listed in Table 1. Gtf2b transcript was used as an internal control to normalize the mRNA levels of each gene.

RNA was reverse transcribed using the Quantitect Reverse Transcription kit (Qiagen). qPCR was performed using SYBR Green technology on a Step One machine (Applied Biosystems). Expression was measured relative to that of GAPDH and significance assessed by performing a one or two tailed t-test on the Ct values. Graphs show mean ± standard deviation.

Total RNA was isolated from GL261 cells using E.Z.N.A.^® Total RNA Kit I (R6834, Omega). Then, the RNA concentration was determined using a NanoDrop One Spectrophotometer (Thermo Fisher Scientific), and reverse transcription was performed to obtain cDNA (RR047A, Takara). Finally, the expression level of ASB3 was determined through RT-qPCR using the Applied Biosystems StepOne machine (Applied Biosystems) and SYBR Green (4913914001, Roche). Primers for β-actin and ASB3 were purchased from Comate Bioscience Co. Ltd., with the following sequences: β-actin-F:5’-GTGACGTTGACATCCGTAAAGA-3’; β-actin-R:5’-GCCGGACTCATCGTACTCC-3’; ASB3-F:5’-TTGAAGTATGGAGCCCAGTTA-3’; ASB3-R:5’-CCAGCAAGCAGGAGATGTG-3’. Relative quantification of the gene expression was carried out via the double delta Ct analysis.

Assay-on-demand kits (Applied Biosystems, Foster City, CA, United States) were used to measure the gene expression of tumoral necrosis factor α (TNF-α), interleukins -1β (IL-1β), −6 (IL-6) and −10 (IL-10), glutathione peroxidase (GPx) and reductase (GSR), superoxide dismutase-1 (SOD-1) and lipoxygenase (Alox5) in myocardial, aortic and gastrocnemius tissues by quantitative real-time polymerase chain reaction (qPCR). The messenger ribonucleic acid (mRNA) levels of NADPH oxidases 1 (NOX-1) and 4 (NOX-4) were also determined in the heart and aorta, and mRNA levels of atrogin-1, Muscle RING-finger protein-1 (MuRF1), Microtubule-associated proteins 1A/1B light chain 3B (LC3b), Insulin growth factor 1 (IGF-1), IGF binding protein 3 (IGFBP3) and Myosin heavy chain isoform I and IIa (MHC-I and MHC-IIa) were determined in the gastrocnemius. Amplification was performed by using the TaqMan Universal PCR Master Mix (Applied Biosystems, Foster City, CA, United States) in a Step One machine (Applied Biosystems, Foster City, CA, United States). 18S and/or hypoxanthine guanine phosphoribosyl transferase (HPRT) were used as housekeeping genes and relative gene expression was determined by the ΔΔC_T method (59 (link)).