Each specific oligonucleotide (NET1-1 and NET1-2) was examined individually and together in the same solution. NET1 mRNA expression was quantified by qPCR and protein expression was examined by Western blot and immunofluorescence. check details Proliferation assay 20 μl of MTS reagent was added to each well of a 96 well plate containing 2 × 104 cells. Treatments were as follows;

10nM scramble siRNA (control), 10nM NET1-1 siRNA, 10nM scramble siRNA + 5 μM LPA and 10nM NET1-1 + 5 μM LPA. After transfection with siRNA, cells were incubated for 24 hours. MTS was then added and the plate was incubated for 2 hours at 37°C and 5% CO2 and absorbance at 492 nm was read using a microplate reader. Migration assay Wound healing migration assays were performed using plastic well inserts (Ibidi, Germany) in 24 well plates. 8 × 104 cells were seeded to each side of a plastic insert inside each well. The following day 10nM NET1-1 siRNA was added with 10nM scramble siRNA acting as a control. Cells were incubated under standard conditions for 24 hours to achieve knockdown of NET1. Inserts were then carefully removed from each well and cells were fed with regular growth medium without siRNA. Wells for LPA treatment were treated with 5 μM in medium. Cells were

observed until they had migrated but not long enough to allow full closure of the gap created by removal of the insert (3 hours). Cells were then fixed using 1:1 methanol acetone and stained with crystal violet. Each well was then photographed at 3 hours and measurements were taken for each condition at three points along the PtdIns(3,4)P2 gap between Selleck Regorafenib mono-layers of cells. All treatment conditions were carried out in triplicate and averages were calculated and recorded as distance in number of pixels across the gap. Comparisons were made between the scramble siRNA and NET1 knockdown

wells. Analysis calculated average migration distances using Image J software (http://​rsb.​info.​nih.​gov/​ij/​). In vitro invasion assay Biocoat Matrigel (BD Biosciences, United Kingdom) invasion chambers were used to investigate and compare the effect of NET1 downregulation on the in vitro invasion of OE33 cells. 1 × 105 cells were seeded to the upper chamber in serum-free medium. Culture medium containing 20% FBS was added to the outer chambers which acted as a chemo-attractant for the cells. The plates were then incubated for 24 hr in a 5% CO2 humidified 37°C incubator. Following incubation, the cells which had invaded the membrane were fixed and stained. The membrane was then removed and mounted on a slide for microscopic assessment. Invasive cells were visualised at 40X magnification and the number of cells in five random fields were counted and an average calculated for each condition. Statistics All experiments were carried out in triplicate unless otherwise stated in results section.