Fig. 4. (A) NHU cells were treated with TZ (1 µM) for the length of time indicated, the medium was changed and the cells were maintained in medium containing PD153035 (1 µM) for 4 days. The RNA was extracted and the RPA was performed and analysed as outlined in Fig. 2. (B) NHU cells were treated for 24 hours with TZ (1 µM) and then maintained in medium in the presence or absence of PD153035 (1 µM) for the times indicated. Uroplakin mRNA expression was quantified by phosphorimager analysis of the RPA hybridisation signal and normalised to the GAPDH signal, to correct for sample loading. Uroplakin expression in TZ-exposed NHU cells treated with PD153035 for 4 days was designated 100%. Diamonds, TZ+PD153035; squares, TZ-PD153035. The data is the mean + s.e.m. of three experiments performed on three independent NHU cell lines.^*P<0.005 TZ compared with TZ+ PD153035. (C) NHU cells were treated in the absence or presence of TZ (1 µM) for 24 hours before the medium was changed and cells were incubated in the presence or absence of PD153035 (1 µM) for 4 days. RPA was performed and quantified as outlined in (A). TZ+PD153035 was taken to be 100% for UPIb. (D) Inhibition of PPAR activation by pre-treatment with the PPAR antagonist GW9662. NHU cells were pretreated for 3 hours with GW9662 at the concentrations indicated, before being exposed to TZ (1 µM) for 24 hours and thereafter to PD153035 (1 µM), all in the continued presence of GW9662. After 4 days, total RNA was extracted and 5 µg were analysed by RPA to assess relative uroplakin mRNA expression, as described in (A). TZ+PD153035 was taken to be 100%.