Figure 3
From: Hnf1b renal expression directed by a distal enhancer responsive to Pax8
CNS1 is specifically activated by ectopic expression of pax8 and pax2 and functions as an enhancer in mammalian renal cells. (a) Reporter constructs used in luciferase assay experiments. The backbone plasmid pGL4.23[luc2miniP] was used either unmodified (Empty-Luc) or modified by insertion of the wild type CNS1 (CNS1-Luc) or CNS1 mutated in the Pax8 binding site (CNS1 mut-Luc) upstream of the minimal promoter. (b) Exogenous pax8 and pax2 are able to activate CNS1 enhancer activation in HEK293 cells. Expression vectors for pax8, pax2, pax8ΔO or pax8VP16 were co-transfected with CNS1-Luc or CNS1mut-Luc, and a control Renilla luciferase vector for normalization. Normalized luciferase activity was determined as the Firefly over Renilla luciferase activity. Fold change is expressed as the ratio of normalized values on the normalized value for CNS1-Luc vector alone. Expression of pax8, pax8ΔO, pax8VP16 and pax2, all result in an increase of reporter activation. In every case, this increase is abolished when the mutant construct, CNS1mut-Luc, is used instead of CNS1-Luc. Combined results from three (pax8, pax8ΔO and pax8VP16) or four (pax2) independent experiments. Statistical significance was determined using One-Way ANOVA followed by Tuckey’s multiple comparison test. P-values are annotated on the figure (c) Pax8 immunofluorescence staining in MDCK and IMCD3 cells and counterstaining of nuclei with DAPI. Pax8 is detected in MDCK and IMCD3 cell nuclei. Scale bar: 50 μm. (d) Analysis of CNS1 activity in luciferase reporter assay in MDCK and IMCD3 cell lines. Histograms show the relative luciferase activities in MDCK and IMCD3 cells transfected with Empty-Luc, CNS1-Luc or CNS1 mut-Luc together with a control Renilla luciferase vector for normalization. Fold change is expressed as the ratio of normalized values on the normalized value for Empty-Luc. Combined results from four independent experiments. Statistical significance was determined using One-Way ANOVA followed by Tuckey’s multiple comparison test. P-values are annotated on the figure.
