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
AQP 0
Direction
Sequence (5`-3`)
Forward
TGTTCTGCAGGTGGCTATG
Reverse
TGCTAGGTTTCCTCGGACAG
Forward
TCATCAGCATCGGTTCTGC
Reverse
CAAGCGAGTTCCCAGTCAG
Forward
TAGCCTTCTCCAGGGCTGT
Reverse
CGTGATCTCATGGAGCAGAG
Forward
GTCACTCTGGGCATCCTCAT
Reverse
CTATTCCAGCACCCAAGAAGG
Forward
GCCCATCATAGGAGCTGTC
Reverse
GGTCAACGTCAATCACATGC
Forward
CCACCCTCATCTTCGTCTTC
Reverse
GTAGAAGAAAGCCCGGAGC
Forward
GTGCTGGCTAGGACAGGAAG
Reverse
CTAGGAGAGGGCCTCCAAGT
Forward
TGCCACCTACCTTCCTGATC
Reverse
GACGGGTTGATGGCATATCC
Forward
TGAGCCTGAATTTGGCAATG
Reverse
CAGCGTGGCAATCACGAGC
Forward
CTCAGTGTCATCATGTAGTG
Reverse
GACTATCGTCAAGATGCCG
Forward
GCACTGGGATGCTGATTGT
Reverse
CCAGCCACGTAGGTGAAGAG
Forward
GACGCTGACGCTCGTCTACT
Reverse
TCTGTGATGACCGCTTTGAG
Forward
GAACCTGTTCTACGGCCAGA
Reverse
GTTCCAGGGTCCAGCTACAA
Forward
ATCATGTTTGAGACCTTCAA
Reverse
CATCTCTTGCTCGAAGTCCA
AQP 1
AQP 2
AQP 3
AQP 4
AQP 5
AQP 6
AQP 7
AQP 8
AQP 9
AQP 10
AQP 11
AQP 12
-Actin
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
UC cell 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.