Molecular analysis

Primer design for quantitative Real Time PCR (qRT-PCR)

The primers used for SYBR green qRT-PCR are mentioned in table 1.2. Some sequences for the primers were taken from previous publications, primer sequences without references were designed in house. The transcript sequences were found using mRNA RefSeq (PubMed, NBCI) database and isoform domains were located using Ensemble. Primers for SYBR Green qPCR were designed so the product would be around 100 nucleotides, and where appropriate they were intron spanning. Primers were designed to be sequences between 18-23 nucleotides in length. Primers were in silico validated via the primer BLAST tool (NCBI) and were checked to be appropriate for PCR use with a GC content of 35–65%, a melting temperature of 60–68o_{C and} minimal or no primer dimer/ secondary structures formation.

Primer validation and measuring primer efficiency

Primers were in silico validated using the primer BLAST tool (NCBI). A dissociation melt curve step was used when performing qRT-PCR to ensure the primers were only amplifying one product. The amplication efficacy was calculated for each set of primers by creating a standard curve via serially diluting cDNA generated using 2 µg RNA starting template. The CT values generated from a qRT-PCR reaction were plotted against the log of the amount of starting

77 genetic material. The slope of this graph was calculated using graphpad prism 6. This number was then used in the ThermoFisher qPCR amplification efficiency calculator to work out the % efficiency and the amplification factor which can be incorporated into delta delta CT method, specific to each primer set when calculated the relative quantification of a certain gene.

Real Time quantitative PCR (qRT-PCR) Analysis

Total RNA was extracted from pelleted fibroblasts using the RNeasy (Qiagen) kit according to the manufacturer’s instructions. RNA was quantified using a Nanodrop 1000 spectrophotomer (Thermo). RNA isolated from normal and diseased bladder urothelium and stromal tissue was provided by Dr Jim Catto. RNA (100 ng) was then reversed transcribed using a high capacity cDNA Reverse Transcription kit (Applied Biosystems), according to the manufacturer’s protocol using a DNA engine Peltier Thermal cycler (MJ Research). Specific mature microRNAs were reverse transcribed using 10 ng of RNA using specific Taqman Reverse Transcription microRNA probe/primers for miR-143, miR-145 and RNU 48 as a control (Applied Biosystems). cDNA was then used in SYBR green or Taqman real time quantitative PCR reaction using a 7900HT fast Real Time-PCR system. Sequences of primers used for SYBR analysis (Sigma) are supplied in table 1.1. Taqman probes for microRNA-143, miR-145, RNU 48 and versican isoforms V0, V1, V2 and V3 were purchased from Applied Biosystems. Quantification was calculated using delta delta CT values and were normalised to endogenous controls U6 (SYBR green), β2macroglobin (B2M) or RNU 48 (Taqman).

qRT-PCR analysis

The quantification of gene expression was calculated via the delta delta CT method (2∆∆CT) (Livak & Schmittgen, 2001). The CT value is the number of thermal cycles at which the amount of fluorescence reaches a detectable threshold. It corresponds to the amount of template at the start of the reaction.

Amplification efficiencies for each set of primers were assessed by using a standard curve of cDNA. 2 µg RNA was reverse transcribed and serially diluted to create 6 standard dilutions, which were used in a qRT-PCR reaction for a particular SYBR green primer. CT values were plotted on a semi-log scale the line of best fit was calculated (Bio-Rad). The efficiency (E) is calculated:

78 Which can be made into a percentage:

Only primer sets with <80% efficiency were used in this study (see appendix). Western Blotting

Cell lysates were prepared by adding 30 µl RIPA buffer supplemented with complete mini protease inhibitor cocktail (Roche) and benzonuclease (Sigma) to cell pellets washed in PBS. Alternatively the RIPA buffer was added directly to the wells to lyse the cells, in order to not lose any extracellular matrix components. The protein concentration was then assessed using a bicinchoninic acid (BCA) assay kit (Thermo) using BSA to generate a standard curve. Absorbance of standards and test samples was measured at 595 nm using a POLARstar Galaxy spectrophotometer (BMG LABTECH).

If appropriate, total protein lysates were treated with chondroitinase ABC (Sigma; 0.003–0.01 units/ml) at 37o _{C for 3 h, or on ice as a control. Total protein (20-30 µg) was resolved by 3–8%} (w/v) SDS-polyacrylamide gel (Life-Technologies) electrophoresis (SDS-PAGE) in Tris-acetate buffer (Life-Technologies). The protein was transferred onto a nitrocellulose membrane (Millipore) by wet transfer or iBlot dry transfer (Invitrogen),blocked with 5% (w/v) milk and 3 % (w/v) bovine serum albumin (BSA) in Tris buffered saline with 0.05% (v/v) Tween 20 (TBS-T) to prevent non-specific binding. The primary antibody was diluted in aforementioned blocking solution and incubated at 4 oC overnight. The membrane was washed 3 times (for 5 min each) with TBS-T, and subsequently incubated with a horseradish peroxidase- conjugated (HRP) secondary antibody diluted 1 in 3000 in the above blocking solution, for 1 h at room temperature. The membrane was then washed 3 times in TBS-T, and developed using enhanced chemoluminescent (ECL) Western Blotting Substrate (Pierce) and HRP activity of the secondary antibody was exposed on an x-ray film (Thermo Scientific) and developed using a Compact X4 Developer (Xograph Imaging Systems).

Nitrocellulose membranes were stripped of antibodies by incubation in 50 ml stripping buffer (62.5 nM Tris, 2% (v/v) SDS, 100 mM β-mercaptoethanol) at 50 o_{C for 30 min. The membranes} were then washed 4 times with TBS-T, and blocked and reprobed as above.

79 Densitometry

The size of detected bands by immunoblotting were quantified used ImageJ analysis software. The scanned blot was converted to an 8- bit image and boxes were drawn, of the same size, around each band assigning them a lane using the ‘plot lanes’ command. The area of each analysed lane was measured, which corresponds to the size of the band. These were adjusted against the size of the loading control bands to calculate the relative protein quantification and the process repeated for each repeat.

Immunocytochemistry

For visualising the α smooth muscle actin stress fibres the fibroblasts were stained with a FITC conjugated anti-alpha smooth muscle actin mouse antibody (clone 1A.4; 1:100). Fibroblasts were seeded on coverslips (see section0) and transfected and/or treated. The coverslips were then removed, and the cells remaining in the culture plate could be harvested and used for molecular analysis. The fibroblasts on the coverslips were washed with PBS twice and subsequently fixed using 100% methanol for 10 min. Fibroblasts were then permeabilised by using 4 mM sodium deoxycholate (diluted in PBS), by washing with it once and then keeping the coverslips in 4 mM sodium deoxycholate for 10 min. Coverslips were then blocked in 2.5% (w/v) BSA in PBS for 30 min before incubating in 25 µl FITC-conjugated αSMA diluted in blocking buffer antibody overnight in the dark at 4 o_{C. After antibody incubation, the coverslips were washed} twice with PBS, keeping the coverslips in the dark to prevent the FITC bleaching. The coverslips were mounted on normal microscope slides using mounting media containing DAPI (vectorshield). The slides were viewed using a Ziess Axioplan 2 fluorescence light microscope at 40x, and imaged using Proplus 7.0.1 image software. The fluorescence intensity was measured using ImageJ software. The background fluorescence was subtracted, pixel intensity measured and normalised to the cell number to give the quantified fluorescent intensity per cell.