Fig. 3. Ribonuclease protection assay (RPA) to quantify the effect of TZ and PD153035 on uroplakin mRNA expression in NHU cells. (A) NHU cells were treated for 24 hours with or without 1 µM TZ, before incubation for 4 days with the EGFR inhibitor PD153035 at the indicated concentrations. Medium containing appropriate inhibitor was replenished every 2 days. Total RNA was extracted and 5 µg were analysed by RPA to assess the relative uroplakin mRNA expression (maximum uroplakin expression assigned 100%), as described in Fig. 2. (B) Concentration-dependent effects of RZ and TZ on uroplakin mRNA expression in EGFR-inhibited NHU cells. NHU cells were pretreated for 24 hours with the indicated concentrations of RZ or TZ, before being incubated in medium containing PD153035 (1 µM). Total RNA was extracted from samples after 4 days and 5 µg were analysed by RPA to assess relative uroplakin mRNA expression, as described in Fig. 2. Maximum uroplakin expression was taken to be 100%. Note that PD153035 alone had no effect on UPII gene expression (B).