Figure 5

Immunoblot of whole-cell lysates from representative NHU cell donors showing effect of P63 siRNA on (a) P63 and KRT13, (b) FOXA1 and (c) GATA3 protein expression, with (+) and without (−) differentiation induction at 48 h. ACTB=β actin loading control. FOXA1 and GATA3 were on the same membrane and normalised to the ACTB shown with FOXA1. (d) Densitometry measurements from immunoblots showing log₍₂₎ fold change of intensity in immunoblotting for three independent donors for P63 and KRT13, and two independent donors for GATA3 and FOXA1 following P63 siRNA, relative to control siRNA. Statistical test performed where material from three donors was measured was a Repeated Measures one-way ANOVA with Greenhous–Geisser correction and Sidak’s multiple comparison post test, with P-values indicated by *P≤0.05, **P≤0.01, ***P≤0.001 and ****P<0.0001. (e and f) RT-qPCR results from NHU cells from three independent donors showing change in abundance of RNA transcript after exposure to P63 siRNA either with or without induction of differentiation for 48 h for (e) urothelial differentiation-associated, and (f) genes associated with P63 motif-containing FAIRE peaks. Log₍₂₎ fold change measured relative to control siRNA with or without differentiation induction. All qPCR transcript relative abundance measurements were normalised internally to GAPDH. Statistics was performed using a two-way ANOVA with Dunnett’s multiple comparison post test, with P-values indicated by *P≤0.05, **P≤0.01, ***P≤0.001 and ****P<0.0001