Detection system

RNA was isolated with TRI Reagent (Sigma Aldrich), and total RNA was isolated following the manufacturer's procedure. For real-time PCR, 500 ng total RNA was reverse transcribed using random hexamer primers, deoxynucleoside triphosphates, MultiScribe reverse transcriptase, and RNase inhibitor (Applied Biosystems, Foster City, CA). cDNA samples were diluted 1:10, and aliquots of 2 μl of the sample cDNA were mixed with SYBR Green JumpStart Taq ReadyMix (Sigma Aldrich), prevalidated primers, and diethylpyrocarbonate-treated water and were measured in duplicate for each sample. Primers used were fatty acid synthase (FAS), SREBP-1c, sterol-CoA desaturase 1 (SCD1), PPARγ, and phosphoenolpyruvate carboxykinase 1 (Pck1). For sequences, see supplementary Table VIII. Expression analysis was performed using the BioRad detection system. Data were normalized to the housekeeping gene 18S.

Total RNA extracted from cells and tissues using the RNAPure Tissue & Cell Kit (DNase I) in accordance with the manufacturer's instructions (CWBIO, Beijing, China). Then, a total of 1 μg RNA was submitted to the reverse transcription of miRNA and mRNA using stem-loop primers and random primers with TaKaRa system (Dalian, China) according to the manufacturer's instructions. Subsequently, the RT-PCR was carried out using a TaqMan Universal Master Mix II kit on a Bio-Rad detection system (Bio-Rad, Hercules, CA). GAPDH and U6 expression levels are served as internal references to normalize mRNA and miRNA expressions, respectively. Primers were listed as follows.
LUCAT1: forward- (F-) 5′-CCTCACAAGAAGCTCACCCA-3′, reverse- (R-) 5′-CAGCATGTAGCCCATGGTAGA-3′; KRAS: F-5′-TAGGCAAGAGTGCCTTGACG-3′, R-5′-CCCTCCCCAGTCCTCATGTA-3′; and GAPDH: F-5′-CCACTAGGCGCTCACTGTTCTC-3′, R-5′-ACTCCGACCTTCACCTTCCC-3′. If the expression was lower than the median value, miR-181c-5p was considered as the low expression and vice versa. Similarly, LUCAT1 was considered to express at a high level when its expression was higher than the median value and vice versa.

To determine the protein expression, RAW264.7 cells were seeded at a cell density of 5 × 10⁴ cells/plate in a 60π plate, and then subjected to pre-treatment for 1 h at 37 °C with and without LPS (1 μg/mL; Sigma-Aldrich) and PUG treatment with 4 and 6 μM for 24 h. After this, RIPA buffer (iNtRON Biotechnology, Incheon, Korea) was added to the cells to lyse and harvest the protein. Then the concentration of protein was estimated by the Pierce™ BCA protein assay kit (Thermo Scientific). To separate the proteins, an equal concentration of protein (10 μg) was loaded in SDS-PAGE. Then the gel was transferred to PVDF membranes via a Semi-Dry Transfer unit (Atto Corporation, Tokyo, Japan). The transferred membrane was then blocked for 1–2 h at room temperature with 5% Bovine Serum Albumin (BSA) and followed by incubation at 4 °C overnight with diluted (1:1000) primary antibodies. The membranes were washed with 1X TBST solution and probed with the horseradish peroxidase-conjugated secondary antibody (1:5000) anti-mouse (cell signaling; cat. No. A90-116P) for β-actin and anti-rabbit (cell signaling; cat. No. A120-101P) to the rest of the proteins at room temperature for 2 h and washed again. Finally, the blots were detected under detection system (Bio-Rad Laboratory), after developing the ECL solution. Protein densitometry was evaluated using the ImageJ software program.

After shrimp tissues were collected at the 12 and 24 hpi time points, total RNA was extracted and subjected to cDNA synthesis by using Superscriptase II Reverse Transcriptase (Invitrogen) and Anchor-dTv primer (Table 1). The cDNA samples were used to quantify the mRNA expression of the target genes by using real-time PCR with KAPA SYBR1 FAST Master Mix (KAPA) and the Bio-Rad detection system. The gene expression levels measured in this study were for the two GLS isoforms (GLS1 and GLS2), IDH1, IDH2, α-KGDH, the WSSV major structural gene VP28 and EF1α. The specific primer sets for each target gene are listed in Table 1. Data values were normalized to EF1α cDNA (internal control) and calculated by the 2^−ΔCT method. Statistically significant differences between groups were analyzed by Student's t-test as described in Tseng et al. (19 (link)).

First-strand cDNA was synthesized by using aliquots of 1 µg of total RNA from cortex and basal ganglia, reverse transcriptase and random primers [7] (link). Specific genes were quantified by real-time PCR using i-Cycler MYIQ system (Bio-Rad, Mississauga, ON). cDNA prepared from total RNA of cultured cells was diluted 1∶1 with sterile water and 5 µl were thereafter used per RT-PCR reaction. Semi-quantitative analysis was performed by monitoring in real time the increase of fluorescence of the SYBR Green dye on a Bio-Rad detection system, as previously reported [57] (link) and expressed as relative fold change (RFC) compared to control. Oligonucleotide primers are provided in Table 1.

The relevant protein levels of TGF-β1, Smad3, P-Smad3, Smad7, α-SMA and collagen-1 in vitro were measured by western blot. Cells incubated and treated in 6-well plates were lysed by 200 μL RIPA lysis buffer containing protease inhibitor (PMSF, 1 mM) and phosphatase inhibitor (TIANDZ 80809-1, 1%) on ice-bath for 30 min. And the lysate was centrifuged at 10000 rpm for 10 min at 4 °C, the supernatant was collected and the proteins were quantified by BCA protein assay kit (Beyotime Biotechnology, China). Membranes were incubated with primary antibodies (anti-TGF-β1, 1:1000, Abcam, ab64715; anti-Smad3, 1:1000, Abcam, ab40854; anti-P-Smad3, 1:400, Bioss, BS-3425R; anti-Smad7, 1:400, Boster, BA1399; anti-α-SMA, 1:400, Boster, BM0002; anti-collagen1, 1:400, Boster, BA0325) overnight at 4 °C and mouse monoclonal anti-β-actin (1:500, Sinipept, 41302M) was used for loading control. After that HRP-goat-anti-mouse antibody (1:1000, World Biotech, WH-004) was incubated with membranes of TGF-β1, α-SMA and β-actin for 1 h at room temperature; HRP-goat-anti-rabbit antibody (1:1000, CW-biotech, cw-0103) with Smad3, P-Smad3, Smad7 and collagen-1 respectively. Immunoblot were visualized with Bio-Rad and recorded by Bio-Rad detection system, and gray density analysis was completed with the Image J program.