Modified eagle s medium

Juvenile greenlip abalone (Haliotis laevigata) were farmed in Tasmania and distressed for a week without feeding in 4000-l tanks filled with filtered sea water at 14–18 °C. Dispase, sodium chloride (NaCl), potassium chloride (KCl), calcium chloride (CaCl₂), CelLytic M reagent, ethidium homodimer, phenazine methosulfate (PMS), 3,3′,5,5′ tetramethylbenzidine (TMB), bovine serum albumin (BSA) and ammonium sulphate were purchased from Sigma-Aldrich, Australia. Phosphate buffer solution (PBS pH 7.4), antibiotic–antimycotic solution (anti-anti) 100 × , fetal bovine serum, KnockOut™ serum replacement, minimum essential medium (MEM) vitamin solution 100 × , minimum essential medium (MEM) amino acid solution 100 × , chemically defined lipid concentrate, (2,3-Bis-(2-Methoxy-4-Nitro-5-Sulfophenyl)-2H-Tetrazolium-5-Carboxanilide (XTT) assay kit, Insulin-Transferrin-Selenium-Ethanolamine (ITS -X) (100 ×), fluorescein isothiocyanate (FITC)-conjugated anti-rabbit antibody from goat and Qubit 2.0 Fluorometric Protein Quantification kit were supplied by Invitrogen (US). F-12 nutrient mixture ham medium, Leibovitz’s L-15, modified Eagle’s medium (MEM) and Dulbecco’s modified Eagle’s medium (DMEM) were supplied by Gibco, Lipimax® by Selbourne Biological Service (Aus), magnesium chloride and magnesium phosphate by MERCK. Rabbit anti-hemocyanin polyclonal antibody was produced in-house.

Human umbilical vein endothelial cells (HUVECs), purchased from Zhong Qiao Xin Zhou Biotechnology (DFSC-EC-01; Shanghai, China), were cultured in 500 mL endothelial cell basal medium containing 25 mL fetal bovine serum, 5 mL endothelial cell growth factor and 5 mL penicillin/streptomycin solution. RCC cell lines A498, 786-O, Caki-1, and 769-P, and human normal kidney cell line HK-2 purchased from ATCC (MA, VA) were cultured in modified Eagle’s medium (Gibco Company, Grand Island, NY) containing 10.0% FBS (Gibco) and 1.0% antibiotics (100 U/mL penicillin and 100 mg/mL streptomycin).
Cells were transfected with plasmids of sh-SNHG12 (short hairpin RNA-SNHG12), oe-SNHG12 (SNHG12 overexpression), sh-KMT2B, oe-E2F1, oe-SNHG12 + sh-E2F1, sh-E2F1 + oe-CEP55, using the Lipofectamine 2000 reagent (Invitrogen, Carlsbad, CA). Corresponding negative control (NC) groups were set up. The plasmids were all constructed by GenePharma Co., Ltd. (Shanghai, China), and the plasmid concentration was 50 ng/mL. The shRNA sequences are shown in Additional file 1: Table S1.

Collagen type I hydrogels were cast using either HUVECs only (100,000 cells/ml) or with co-cultures of HBMSCs and HUVECs. Four cell ratios were tested by increasing the number of HUVECs in the gels and keeping the number of HBMSCs constant. In all hydrogels 200,000 HBMSCs were used and HUVECs were used between 100,000 cells/ml and 400,000 cells/ml. HBMSC only hydrogels were also cast and used as controls (200,000 cells/ml). Briefly, 800 µl of rat tail collagen type I (2.05 mg/ml, FirstLink, UK) was mixed with 100 µl of 10× Modified Eagle׳s Medium (Gibco, UK ) and was neutralised using drop wise addition of 5 M and 1 M NaOH solution. The cells (in 100 µl medium) were then mixed with the collagen solution and cast in a 12-well plate. Hydrogels were cultured in either EGM (HUVEC only) or in a 1:1 mixture of DMEM and EGM (co-cultures). Endothelial cell only cultures were cultured for 1 week and 2 weeks and co-cultures were cultured for 1 week only (due to excessive contraction of the gels). Hydrogels were analysed for VEGF protein levels using ELISA. VEGF receptors were quantified using flow cytometry and CD31 immunofluorescence was used to test cell morphology and aggregation.

Faecal samples were prepared as 10% suspensions in a balanced salt solution (Modified Eagles Medium, Gibco, Life Technologies, Paisley, UK). Total nucleic acid was extracted from a 200 µl aliquot of faecal suspension using the QIAxtractor automated nucleic acid extraction platform (QIAGEN Ltd., Crawley, UK), VX Reagent & Plasticware kit (QIAGEN). Complementary DNA (cDNA) was generated by reverse transcription as follows: a 40 µl aliquot of extracted nucleic acid was heat denatured at 95°C for 5 minutes then quenched on ice. Denatured nucleic acid was mixed with 30 µl reverse transcription reaction mix, prepared as follows: 40 mM random hexamers (Invitrogen, Paisley, UK), 10 mM Tris, pH 8.0, 50 mM HCl, 5 mM MgCl₂, 1 mM of each dNTP (Invitrogen), 400 U of MuMLV reverse transcriptase (Invitrogen). Reverse transcription was performed at 37°C for 1 hour, and the reaction was terminated by incubation at 95°C for 5 minutes, and then cooled on ice.