Supplementary Methods
Table 1. Clinico-pathological characteristics of the study cohort
Parameter / n (%)Total number of patients / 94 (100.0)
Sex
Female patients / 17 (18.1)
Male patients / 77 (81.9)
Age
Median age at surgery (years) / 68
Range age (years) / 40 – 90
WHO Grading
WHO 1973 G1 / 15 (16.0)
WHO 1973 G2 / 30 (31.9)
WHO 1973 G3 / 49 (52.1)
TNM Staging
pTa / 43 (45.7)
pT1 / 30 (31.9)
pT2 / 21 (22.4)
Specimens
TURB specimen / 73 (77.6)
Radical CX specimen / 21 (22.4)
CX cystectomy, TURB transurethral resection, WHO World Health Organization
Table 2. RT-PCR oligonucleotide primers
Gene / Direction / Sequence (5`-3`)AQP 0 / Forward / TGTTCTGCAGGTGGCTATG
Reverse / TGCTAGGTTTCCTCGGACAG
AQP 1 / Forward / TCATCAGCATCGGTTCTGC
Reverse / CAAGCGAGTTCCCAGTCAG
AQP 2 / Forward / TAGCCTTCTCCAGGGCTGT
Reverse / CGTGATCTCATGGAGCAGAG
AQP 3 / Forward / GTCACTCTGGGCATCCTCAT
Reverse / ctattccagcacccaagaagg
AQP 4 / Forward / GCCCATCATAGGAGCTGTC
Reverse / GGTCAACGTCAATCACATGC
AQP 5 / Forward / CCACCCTCATCTTCGTCTTC
Reverse / GTAGAAGAAAGCCCGGAGC
AQP 6 / Forward / GTGCTGGCTAGGACAGGAAG
Reverse / CTAGGAGAGGGCCTCCAAGT
AQP 7 / Forward / TGCCACCTACCTTCCTGATC
Reverse / GACGGGTTGATGGCATATCC
AQP 8 / Forward / TGAGCCTGAATTTGGCAATG
Reverse / CAGCGTGGCAATCACGAGC
AQP 9 / Forward / CTCAGTGTCATCATGTAGTG
Reverse / GACTATCGTCAAGATGCCG
AQP 10 / Forward / GCACTGGGATGCTGATTGT
Reverse / CCAGCCACGTAGGTGAAGAG
AQP 11 / Forward / GACGCTGACGCTCGTCTACT
Reverse / TCTGTGATGACCGCTTTGAG
AQP 12 / Forward / GAACCTGTTCTACGGCCAGA
Reverse / GTTCCAGGGTCCAGCTACAA
-Actin / Forward / ATCATGTTTGAGACCTTCAA
Reverse / CATCTCTTGCTCGAAGTCCA
UC line culture and maintenance
The three established urothelial carcinoma (UC) cell lines RT4, RT112 and T24 were obtained from the Health Protection Agency Culture Collection (HPACC; Porton Down). Prior to use, all UC cell lines were genotyped to verify their pedigree (see “UC line authentication” below). Cells were cultured in ‘DR’, a 50:50 (v/v) mixture of Dulbecco’s modified Eagle medium (DMEM) and Roswell Park Memorial Institute 1640 (RPMI) medium (Sigma). This was supplemented with 5% Fetal calf serum and 1% L-Glutamine (DR/5%FCS/1%L-G). Cells were maintained in a humidified atmosphere at 37°C in 10% CO2 in air.
UC line characteristics
RT4 is a well differentiated human UC cell line derived from G1/Ta recurrent papillary tumour (Rigby and Franks, 1970, Brit J Cancer 24:746), RT112 is moderately-differentiated cell line derived from G2/T1 tumour (Masters et al., 1986, Cancer Res 46:3630) and T24 (EJ) is a poorly differentiated and highly anaplastic human UC cell line that is derived from G3/ T2 minimum tumour (Marshal et al., 1977, JNCI 58:1743).
UC line authentication
UCcell lines were genotyped using a PCR-based short tandem repeat (STR) analysis system to verify cell line pedigree using the Powerplex® 1.2 system (Promega) that allows identification of 9 independent loci (Penta E, D18S51, D21S11,TH01, D3S1358, FGA, TPOX, D8S1179 and vWA) from genomic DNA.Genomic DNA was isolated using a DNeasy Blood and Tissue Kit (Qiagen). PCR reactions were performed as recommended by the manufacturer (Promega) in a GeneAmp® PCR system 9700 (Applied Biosystems). Following PCR, samples were injected into a Beckman CEQ 8000 fragment analyzer capillary electrophoresis system along with an allelic ladder (Promega). Results were analysed using GeneMapper® 4.0 software (Applied Biosystems) and the STR profile for each sample was compared to that found on the Health Protection Agency Culture Collection (HPACC) or American Type Culture Collection (ATCC) websites.