Cells were cultured in T-75 flasks maintained at 37°C in a humidified atmosphere of 5% CO2. Het1a required a supporting layer composed of extracellular matrix proteins for subculture. Flasks were coated with 0.01 mg/ml bovine serum albumin, 0.01 mg/ml fibronectin and 0.03 mg/ml bovine type I collagen and were incubated
overnight at 37°C in 5% CO2. Het1a was cultured in BEBM medium containing BPE 0.4%, insulin 0.5 ml, hydrocortisone 0.5 ml, gentamicin/amphotericin 0.5 ml, retinoic acid 0.5 ml, transferring 0.5 ml, triiodothyronine 0.5 ml, epinephrine 0.5 ml and hEGF 0.5 ml (Lonza Clonetics, Walkersville, USA). Flasks were maintained at 37°C in a humidified atmosphere of 5% CO2. RNA extraction and qPCR RNA extraction was carried out using TRIzol™ reagent (Sigma Aldrich, Ireland) under standard {Selleck Anti-infection Compound Library|Selleck Antiinfection Compound Library|Selleck Anti-infection Compound Library|Selleck Antiinfection Compound Library|Selleckchem Anti-infection Compound Library|Selleckchem Antiinfection Compound Library|Selleckchem Anti-infection Compound Library|Selleckchem Antiinfection Compound Library|Anti-infection Compound Library|Antiinfection Compound Library|Anti-infection Compound Library|Antiinfection Compound Library|Anti-infection Compound Library|Antiinfection Compound Library|Anti-infection Compound Library|Antiinfection Compound Library|Anti-infection Compound Library|Antiinfection Compound Library|Anti-infection Compound Library|Antiinfection Compound Library|Anti-infection Compound Library|Antiinfection Compound Library|Anti-infection Compound Library|Antiinfection Compound Library|Anti-infection Compound Library|Antiinfection Compound Library|buy Anti-infection Compound Library|Anti-infection Compound Library ic50|Anti-infection Compound Library price|Anti-infection Compound Library cost|Anti-infection Compound Library solubility dmso|Anti-infection Compound Library purchase|Anti-infection Compound Library manufacturer|Anti-infection Compound Library research buy|Anti-infection Compound Library order|Anti-infection Compound Library mouse|Anti-infection Compound Library chemical structure|Anti-infection Compound Library mw|Anti-infection Compound Library molecular weight|Anti-infection Compound Library datasheet|Anti-infection Compound Library supplier|Anti-infection Compound Library in vitro|Anti-infection Compound Library cell line|Anti-infection Compound Library concentration|Anti-infection Compound Library nmr|Anti-infection Compound Library in vivo|Anti-infection Compound Library clinical trial|Anti-infection Compound Library cell assay|Anti-infection Compound Library screening|Anti-infection Compound Library high throughput|buy Antiinfection Compound Library|Antiinfection Compound Library ic50|Antiinfection Compound Library price|Antiinfection Compound Library cost|Antiinfection Compound Library solubility dmso|Antiinfection Compound Library purchase|Antiinfection Compound Library manufacturer|Antiinfection Compound Library research buy|Antiinfection Compound Library order|Antiinfection Compound Library chemical structure|Antiinfection Compound Library datasheet|Antiinfection Compound Library supplier|Antiinfection Compound Library in vitro|Antiinfection Compound Library cell line|Antiinfection Compound Library concentration|Antiinfection Compound Library clinical trial|Antiinfection Compound Library cell assay|Antiinfection Compound Library screening|Antiinfection Compound Library high throughput|Anti-infection Compound high throughput screening| conditions. Quantitative PCR was carried out by the SyBr Green method using the Rotor-Gene™ 3000A Real buy LBH589 Time Thermal Cycler and the Rotor-Gene™ 6 software package. Specifically designed primers for NET-1 were purchased from Qiagen (Crawley, West Sussex, UK) and GAPDH was used as an endogenous control. Western blot Following LPA stimulation or siRNA treatment, cells were lysed and total protein was analysed by immublot using SC-50392 (Santa Cruz, United States) NET1 specific rabbit IgG monoclonal antibody. Immuno-fluorescence 2 × 104 cells were seeded into 8
well chamber slides, treated with Vistusertib mouse either NET-1 specific siRNA or scramble siRNA and incubated at 37°C for 24 hours with 5% CO2. Immuno-fluorescence was measured using SC-81333 (Santa Cruz, United States) NET1 specific mouse IgG monoclonal antibody and a FITC labelled Protirelin secondary antibody. Optimisation of LPA treatment by dose/response In order to determine optimal treatment conditions for LPA in OE33 and het1a cell lines a dose/response experiment was performed. Cells were treated with 1, 5, 10 and 20 μl LPA and. NET1 mRNA expression was quantified by qPCR and protein expression was examined by Western blot. Gene knockdown by siRNA Two siRNA duplexes were designed and synthesised for silencing NET1 (Qiagen Inc. CA, USA). The duplexes were termed: NET1-1 (sense, 5′- GGA GGA UGC UAU AUU GAU A-3′;
antisense, 5′- UAU CAA UAU AGC AUC CUC C-3′) and NET1-2 (sense, 5′- GGU GUG GAU UGA UUG GAA A- 3′; antisense, 5′ UUU CCA AUC AAU CCA CAC C-3′). A chemically synthesized non-silencing siRNA duplex (sense, 5′-UUC UCC GAA CGU GUC ACG U-3′; antisense, 5′-ACG UGA CAC GUU CGG AGA A-3′) that had no known homology with any mammalian gene was used to control for non-specific silencing events. 4 × 105 OE33 cells were added to each well of a 6-well plate containing 2 ml growth media and were incubated under the standard conditions of 37°C and 5% CO2 in a humid incubator for 24 hr. After 24 hrs the siRNA-containing culture medium was aspirated and 1.9 ml of new medium was added to each well. 1 μl (0.3 μg, 10nM), 5 μl (1.5 μg, 50nM), 17 μl (5 μg, 170nM) and 25 μl (7.