Fig 9. DAPT prevented MM cell differentiation in E12.5 embryonic kidneys during organ culture and maintained NPC in aggregates derived therefrom.
(A) E12.5 embryonic kidneys were treated with various inhibitors for 7 days in organ culture. qRT-PCR results show the effect of Notch signal inhibitor (DAPT) in suppressing the expression of MM differentiation marker genes (Podxl1, Nkcc2 and Slc5a1, but not Slc12a3). No effects were seen with Wnt signal inhibitor (IWP2), GSK3β inhibitor (BIO), JNK inhibitor (SP600125) or BMP signal inhibitor (Dorsomorphin). Data were normalized by Gapdh expression levels and presented as fold changes from DMSO treated embryonic kidneys as control. (N.D: not detected). (B) E12.5 embryonic kidneys were treated with different inhibitors for 7 days in organ culture. Cells were then dispersed from these embryonic kidneys to reconstitute aggregates and cultured for another 7 days without respective inhibitors. Immuno-staining of representative aggregates for NPC marker, Six2 (green), and UB marker, DBA (red), shows the presence of Six2+-NPC only in aggregates derived from DAPT-pretreated embryonic kidneys. (Scale bars = 500 μm)