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. Author manuscript; available in PMC: 2013 Apr 25.
Published in final edited form as: Nat Cell Biol. 2012 Apr 22;14(5):477–487. doi: 10.1038/ncb2490

Figure 4.

Figure 4

Acute loss of Id genes in NSCs impairs cell adhesion and induces bHLH-mediated transcription of Rap1GAP. (a) Immunofluorescence staining for vinculin (red) and F-actin (phalloidin, green) in NSCs treated with 4-OHT or vehicle for 18 h. (b) Cell adhesion on ECM components of NSCs treated with 4-OHT for 48 h. Left, upper panels, bright-field images of NSCs treated with vehicle or 4-OHT 1 h after plating on fibronectin-coated plates. Left, middle panels, bright-field images of NSCs remaining adherent after washes. Left, lower panels, crystal violet staining of NSCs remaining adherent after washes. Right, colorimetric quantification of NSCs adherent to laminin or fibronectin. Data represent the means; n = 8 from two independent experiments, each performed in quadruplicate. P = 5.401 × 10−8 (adhesion to laminin); P = 2.383 × 10−7 (adhesion to fibronectin). Asterisks indicate statistical significance. (c) Rap1GAP promoter (upper scheme) and Rap1GAP promoter/luciferase construct (lower scheme). Squares indicate E-boxes. Arrows indicate primers used to amplify the E-box-containing regions in ChIP assay. 2 and 3 are regions in the Rap1GAP gene that were not bound by E47. (d) Crosslinked chromatin prepared from NSCs transfected with E47 was incubated with IgG or anti-E47 antibody. Immunoprecipitates were amplified by PCR and analysed by agarose gel electrophoresis (left panel) or qPCR (right panel). Fold enrichment is relative to background chromatin pulled down from IgG immunoprecipitation. Data represent the means ± s.e.m.; n = 3 independent experiments, each carried out in triplicate; P = 1.400× 10−5. The asterisk indicates statistical significance; n.s., not significant. (e) Rap1GAP mRNA induction as a function of E47 in NSCs. Data represent the means; n = 6 from two independent experiments, each performed in triplicate. (f) Western blot using antibodies against Rap1GAP, E47 and vinculin in NSCs transfected with pcDNA–E47 or the empty vector. (g) NSCs were transfected with pGL vector or pGL–Rap1GAP–2533 in the absence or in the presence of increasing amounts of pcDNA3.1–E47. Data represent the means; n = 6 from two independent experiments, each performed in triplicate. (h) NSCs were transfected with pGL vector or pGL–Rap1GAP–2533 and treated with 4-OHT for 72 h. Data represent the means; n = 6 from two independent experiments, each performed in triplicate. P = 0.0029. RLU is relative Firefly luminescence units normalized to Renilla luminescence. The asterisk indicates statistical significance. Uncropped images of blots are shown in Supplementary Fig. S6.