Tiempo de almacenamiento (meses)

2.6.1 Plasmid preparation

Plasmids used in reporter assays are summarised in Table 2.4. The plasmids were transformed into JM109 cells (Promega, USA) plated on agar plates; single colonies were selected and grown in 1 mL of L-broth containing 50 g/mL ampicillin starter culture at 37 ºC overnight with shaking. The starter cultures were inoculated into 100 mL L-broth and were incubated overnight with shaking at 37 ºC (200 mL in total for each plasmid). The plasmid DNA was extracted from the bacterial culture using a Plasmid Maxi Kit

Primer Primer sequence

44 (Qiagen, USA) according to the manufacturer’s instructions. Plasmid DNA pellets were resuspended in 100 µL of Tris/EDTA (TE) buffer. The plasmid DNA was then quantified using a NanoDrop® ND-1000 spectrophotometer (NanoDrop Technologies, USA). The DNA was stored at -20 ºC.

Plasmid Description Source

pGL3 Control

SV40 promoter and enhancer driving luciferase expression with ampicillin resistance gene

Promega

pXPG ITGA2 874 bp of human ITGA2 promoter (-791 to +83) with ampicillin resistance gene

Cloned by Alison West

pCpGL ITGA2 874 bp of human ITGA2 promoter (-791 to +83) with zeocin resistance gene

Cloned by Annabel Short

RcCMV Contains neomycin and ampicillin

resistance gene Invitrogen

pCMV-Flag SNAIL WT

800 bp of Human SNAII gene with

kanamycin resistance gene Addgene

pCMV6 TWIST1 884 bp of human TWIST1 gene with

ampicillin resistance gene Origene

pXPG ITGA2-E- Boxm

As for pXPG ITGA2 with putative E-Box site mutated with ampicillin resistance gene

Refer to Chapter 5

pXPG ITGA2 Del1 652 bp of human ITGA2 promoter (-569

to +83) with ampicillin resistance gene Refer to Chapter 5

pXPG ITGA2 Del2 353 bp of human ITGA2 promoter (-270

45 EF1α-Sp1Neo Sp1 cDNA with neomycin resistance gene

Professor Merlin Crossley, Ref Crossley et al. 1995 Table 2.4 Plasmids used in transient transfection

2.6.2 Restriction enzyme digests

Restriction enzyme digestions were used to confirm the identity of the plasmids. In general, the concentration of DNA used was between 100 ng and 1 g. The digestion reaction consisted of the 1X recommended buffer (New England Biolabs, USA), 100 µg/mL BSA (New England Biolabs, USA), 1 L of enzyme (20 U/L, New England Biolabs, USA) and MilliQ® water to a total volume of 20 µL. The samples were incubated at the optimal temperature of the enzyme overnight, then analysed by agarose gel electrophoresis.

2.6.3 Methylation of vector

CpG methyltransferase (M.SssI) (New England Biolabs, USA) was used to methylate the pCpGL ITGA2 vector. Approximately 1 µg of vector was methylated with 1/10th volume of NEB2, 1/50th volume of 32 mM SAM and 100 U of M.SssI. The reaction was incubated for 4 hours at 37 ºC. This was followed by an inactivation step at 60 ºC for 20 minutes. To confirm methylation, the vector was digested with AciI methylation sensitive enzymes.

2.6.4 Transfection of prostate cancer cell lines

LNCaP and/or PC3 cells (2 x 106 cells) in 500 µL of PBS were used for each transfection. Transfections were conducted in duplicate. Purified plasmid DNA was added to a Gene Pulser® electroporation cuvette (4 mm; BioRad, USA) along with 500 µL of cells and then electroporated at 300 V with a capacitance of 500 µF using a BioRad Gene Pulsar® PLUSTM electroporator unit. Next, 1 mL of medium was added into each cuvette and the cells were allowed to recover for 5 minutes at room temperature. Cells were removed from the cuvette with a sterile Pasteur pipette and duplicate transfections

46 were combined into a flask containing 7.5 mL medium. The cells were allowed to recover for 24 hours.

2.6.5 Preparation of cell lysates

Following appropriate treatment, the transfected cells were harvested in PBS using a cell scraper (TPP®, Switzerland) and then centrifuged at 500 g for 5 minutes to pellet the cells. The supernatant was discarded and the cell pellets were resuspended in 100 µL of 1X cell lysis buffer (Promega, USA). The cell lysates were subjected to a freeze thaw cycle by placing them at -80 ºC for a minimum of 15 minutes and then returning them to room temperature. This was followed by vortexing for 15 seconds and then centrifuging at 10000 g for 15 seconds. The supernatant containing cellular proteins was removed and an aliquot of the lysate was diluted 1:10 to measure protein concentration using a Bradford assay (Section 2.6.6). The cell lysate was stored at -80 ºC, until analysis.

2.6.6 Bradford assays

Bradford protein assay was used to ascertain the concentration of proteins in cell lysates prepared in Section 2.6.5. A standard curve created using bovine serum albumin (BSA; New England Biolabs, USA) was used to determine the concentration of the protein. The Protein Assay Dye Reagent Concentrate (Bio-Rad, USA) was diluted 1:5 and 1 mL was added to 10 µL of each standard or protein extract, mixed well and allowed to stand at room temperature for 5 minutes. The absorbance at 595 nm was measured for each standard relative to a blank using the BioSpec-mini spectrophotometer (Shimadzu Corporation, USA). Concentrations of samples were determined using standard curves generated using BSA standards.

2.6.7 Luciferase assays

The activity of the integrin promoter cloned into reporter plasmids (Section 2.4) was determined using a Luciferase Assay System (Promega, USA). Protein extracts (30 µg) were added into each well of a 96 well plate in triplicate for each sample. Then, 100 µL luciferase assay reagent (Promega, USA) was added into each well. Luciferase activity

47 was measured using a VeritasTM Microplate Luminometer (Turner Biosystems, USA) program with 2 seconds integration.