Lipofectin reagent

Monoclonal anti-panRas antibody (Ab-3) was purchased from Calbiochem (EMD Biosciences, an Affiliate of Merck KGaA, Darmstadt, Germany); The antibodies against Ki-Ras (sc-521), anti-ERK, phospho-ERK, anti-cyclin A, anti-p53, anti-cyclin D1, anti-p27 and anti-p53 were from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The anti-β- actin was from Sigma–Aldrich (St. Louis, MO, USA). The peroxidase-conjugated (HRP) anti-rabbit, anti-mouse secondary antibodies, nitrocellulose membrane PROTRAN and the ECL detection system were from Amersham-Pharmacia (Biothec, UK Limited). Fetal bovine serum (FBS), trypsin–EDTA, and penicillin/streptomycin solutions were purchased from HyClone Europe Ltd. (Cramlington, UK); Dulbecco’s Modified Eagle’s Medium (DMEM) and Lipofectin reagent were from GIBCO BRL, Life Technologies (Carlsbad, CA, USA). All other reagents were purchased from Sigma Aldrich (Milano, Italy).

Lewis lung cancer (LLC) cells originated as a spontaneous carcinoma of the lung in a C57BL/6 mouse [17] (link), [18] (link). We established LLC cells transfected with the human IL-6 gene (LLC-IL6), which has a potent ability to induce cancer cachexia in mice, as previously described [12] (link). Briefly, human IL-6 cDNA was introduced into Sal I site of the eukaryotic cDNA expression vector BMGNeo, and was transfected into LLC cells using the Lipofectin reagent (Gibco BRL, Gaithersburg, MD) according to the manufacturer’s instructions [12] (link), [19] (link). Cells were maintained in Iscove’s Modified Dulbecco’s Medium (IMDM, Wako, Osaka, Japan) with 20% fetal bovine serum, penicillin and streptomycin (100 U/mL and 100 µg/mL, respectively).

Cell culture systems were performed as previously described (Wu et al. 2016b (link)). The cell bank of Taipei Veterans General Hospital (Taiwan) supplied the human gastric cancer cell lines, AGS (moderately differentiated gastric adenocarcinoma) and MKN 45 or SCM-1 cells (poorly differentiated gastric adenocarcinoma). Cells were grown in RPMI medium supplemented with 10% FBS, 100 U/ml penicillin, and 100 mg/ml streptomycin (complete medium) at 37 °C in a humidified incubator with 5% CO₂. During experiments, cells were plated in six-well plates cultured with serum-free medium (starved medium) overnight and then treated with drugs (honokiol or melatonin) with various concentrations for various time intervals. In some experiments, transfection of cancer cells was performed using a Lipofectin reagent (Invitrogen) according to the manufacturer’s instructions. The efficiency of transfection (~ 90%) was determined using an equal amount of a plasmid that encoded the green fluorescent protein under the cytomegalovirus promoter.

Small interference RNAs (siRNA) design primers, preparation of fungal protoplast and siRNA delivery were performed according to Hanano et al. (2018a) (link). In brief, delivery of siRNA to protoplasts was done in sterile 1.5 mL tubes. A total of 10 μL of siRNA primer (100 nM) was mixed with an equal volume of Lipofectin reagent (Invitrogen Life Technologies, United Kingdom) and kept for 15 min at 20°C. A volume of 50 μL of protoplasts was added, gently mixed and incubated at 20°C for 24 h to allow transfection to proceed. The transfected protoplasts were therefore inoculated in 10 mL of PD medium with 1.2 M of sorbitol for 7 days at 28°C in the dark. All experiments were performed using three biological replicates.

Cancer cells AGS, SCM1, and MKN45 were transfected with shRNA (National RNAi Core Facility Platform, Taipei, Taiwan) or overexpressed plasmid 1 μg/ml pcDNA (Genome Research Center, National Yang-Ming University) using a Lipofectin reagent (Invitrogen), in accordance with the manufacturer’s instructions.

Gene knockdown was conducted with small interfering RNA (siRNA) against the pgc-1α gene. Multiple siRNA sequences were synthesized form by GE Healthcare (GE Healthcare, Chicago, IL, USA). All siRNAs were dissolved in an isotonic RNAi buffer (100 mM potassium acetate; 30 mM Hepes-KOH; 2 mM magnesium acetate; 26 mM NaCl, pH 7.4, at 37 °C). The siRNA against pgc-1α as following blow:
pgc-1αSequenceSequence 15′-CGGUGGAUGAAGACGGAUU-3′Sequence 25′-CAAUGAAUGCAGCGGUCUU-3′Sequence 35′-GAACAAGACUAUUGAGCGA -3′Sequence 45′-AUUCAAACUCAGACGAUUU-3′Pooled siRNAs were mixed with Lipofectin reagent (1:1, 18292-011, Invitrogen, Carlsbad, CA, US) and microinjected bilaterally into the CA3 region of the hippocampus 24 h before the microinjected administration of KA. The pooled non-targeting siRNAs containing four scramble sequences were used as control siRNA.