Positive control (ureter) pT2 G3 UBC(AQP3-negative)

Internal positive control

50µm

Suppl. Figure S1: AQP3 immunoperoxidase labelling: positive controls. Sections of normal urothelium within the specimens served as internal controls. In addition, positive control tissues of normal human urothelium known to express the antigen and negative controls in which the primary antibody was omitted were included in all experiments.

Section of normal urothelium (internal positive control)

Papillary low-grade tumour

(AQP3 +ve)

Suppl. Figure S2: AQP3 immunoperoxidase labelling. Intense, homogeneous AQP3 expression in >75% of a papillary low-grade (pTaG1) tumour. Adjacent normal urothelium showing expression in basal and intermediate, but not in superficial cells. No immunoreactivity was detected in the lamina propria, smooth muscle, or endothelium

25µm

AQP4, +ve control

AQP4

AQP4, +ve control

AQP4, pT1G2

AQP4, pT2G3

Suppl. Figure S3: AQP4 immunoperoxidase labelling. AQP4 was found to be expressed in normal human urothelium but not in UC. Positive controls (human ureter) known to express AQP4 were included in all experiments.

25µm

pT1 G2

+ve controle (ureter) pT2 G3

-ve control (ureter)

25µm

Suppl. Figure S4: AQP7 immunoperoxidase labelling. AQP7 was expressed by all tumour specimens irrespective of grade and stage. Whereas AQP7 localized to the nucleus and the cytoplasm in normal human urothelium and superficial low-grade UC, intense labeling of the nuclei was found in muscle-invasive high-grade UC. Negative controls in which the primary antibody was omitted as well as positive controls known to express the antigen were included in all experiments.

+ve control (human liver)

-ve control (human liver)

human ureter

pTa G1

Suppl. Figure S5: AQP9 immunoperoxidase labelling. AQP 9 was shown to be intensely expressed by human liver (positive control), but no immunoreactivity was detected in ureter and UC specimens.

25µm