Figure 3.

Notch downregulation is necessary to achieve differentiation of human renal progenitors into the podocyte lineage. (A): Sustained Notch protein expression in renal progenitors as obtained following infection with a vector leading to the N1ICD, N2ICD, or N3ICD and the GFP to be simultaneously coexpressed. Cells infected with the empty vector (mock) express GFP but do not express the NICDs. Uninfected cells are shown for comparison. One representative of 10 independent experiments is shown. (B): Downregulation of the Notch pathway activity following differentiation of renal progenitors toward the podocyte lineage is rescued by infection with vectors expressing N1ICD, N2ICD, or N3ICD as demonstrated by coinfection with a reporter vector for the RBP-J transcriptional response element. Results are expressed as mean ± SEM fold increase of luciferase activity in renal progenitors coinfected with a reporter vector for the RBP-J and the vectors expressing N1ICD, N2ICD, or N3ICD in comparison with the empty vector before (control) or after their differentiation toward the podocyte lineage (VRADD) as obtained in at least four independent experiments. b versus a, c, d, e, f, g, h: p < .05; a versus c, e, g: p < .05; a versus d, e versus f, and g versus h: NS. (C): Analysis of nephrin mRNA fold increase after VRADD culture in renal progenitors infected with an empty vector (mock) and with vectors expressing N1ICD, N2ICD, or N3ICD. Results are expressed as mean ± SEM obtained in four separate experiments. (D): Effect of infection with mock vector and vector expressing N1ICD, N2ICD, or N3ICD on cell number after 48 hours of culture in VRADD medium. Results are expressed as mean ± SEM of triplicate assessment as obtained in four separate experiments (*, p < .05). Abbreviations: GFP, green fluorescent protein; RBP-J, recombination signal-binding protein-J; RLU, relative luminescence unit; NICD, Notch intracellular domain; NS, not significant; VRADD, vitamin D₃, retinoic acid and dexamethasone-supplemented DMEM/F12.