Fig. 3

FZD1 knockdown in N2a cells and AHP isolated from adult mouse brain. a qRT-PCR from total RNA isolated from N2a cells 48 h after transfection with retroviral vectors expressing shRNAs (shC or shFZD1) and the fluorescent protein ZsGreen (ZsG). FZD1 mRNA levels were normalized to GAPDH mRNA and expressed relative to shC. Bars represent mean ± S.E. *p < 0.05, Student’s t-test (N = 7 independent experiments). b Immunoblot analysis of FZD1 and β-actin in total protein extracts from N2a cells 48 h after transfection with the retroviral vectors. Lines at the right indicate molecular weight standards: 70 kDa (top) and 40 kDa (bottom). c Immunofluorescence analysis of FZD1 and SOX2 in AHPs 96 h after transfection with retroviral vectors expressing shC or shFZD1. Arrows indicate transfected cells (ZsG+). Scale bar: 20 μm. d Higher magnification of the cells indicated by dotted lines in top panels. Scale bar: 20 μm. e qRT-PCR quantification of the mRNA levels of NeuroD1 and Prox1 in N2a cells 48 h after transfection with shC or shFZD1. mRNA levels were normalized to GAPDH and expressed relative to shC (dotted line). Bars represent mean ± S.E. *p < 0.05, Student’s t-test (N = 3 independent experiments). f Immunoblot analysis of β-catenin and α-tubulin used as loading control, in total protein extracts from AHP cells 48 h after transduction with retrovirus expressing shC or shFZD1. Lines at the right indicate molecular weight standards: 90 kDa (top) and 55 kDa (bottom)