Fig. 7

Klf4 increases Stau1 occupancy on target mRNAs in cortex ex vivo. a (Left) NPCs were transfected with pEGFP-Control, pEGFP-Klf4, or pEGFP-∆C vectors and grown for 0.2 or 2 days in N2 medium. Immunostaining to detect Tuj1 (red) or GFP (green) expression. DAPI (blue). Scale bar, 50 μm. (Right) Quantification of results shown at right. b qPCR analysis of indicated transcripts in NPCs transfected with pEGFP-Control, pEGFP-Klf4, or pEGFP-∆C constructs and cultured in undifferentiation (Un) conditions or for 2 days of differentiation (2D). a, b Data are presented as mean ± SD. ANOVA tests were performed to calculate significance (*P < 0.01, **P < 0.001). See Supplementary Table 4. c, d NPCs transfected with full-length (c) or ∆C Klf4 (d) were treated with actinomycin D for indicated times and mRNAs were prepared for RNA stability assay (n = 3). Results are normalized to control NPCs at 0 h. e Schematic showing the modified ex vivo PAR-CLIP protocol. Green images indicate GFP-labeled control, full-length Klf4 or ∆C expression in electroporated embryo brain. f PAR-CLIP-qPCR analysis in cortices electroporated with pEGFP-Control, pEGFP-Klf4, or pEGFP-∆C constructs and grown in ex vivo condition for 2D. See also Supplementary Fig. 13c