Figure 1
Generation of novel Hh-pathway active neurospheres from mouse cerebellum. (A) Neurosphere formation assay from P7 cerebellar explants grown in different conditions, as indicated. Data obtained by three independent experiments are reported as means +/− SD. (***p < 0.001). GF: EGF + bFGF cocktail. (B) Representative H&E staining of 1 week old S-cNS and GF-cNS of different sizes. Scale bar, 20 μm. (C) Frequency distribution analysis of the biggest diameter in S-cNS and GF-cNS ( ~100 spheres per sample). (D) Western Blot (WB) analysis comparing GLI1 and N-MYC protein expression in standard GCPs cultures and neurosphere cultures (grown as indicated), separated by an empty lane (indicated with/). Blots were probed for β-actin as a loading control. (E) RT-PCR quantification of known SHh target genes in P7 cerebellar extracts (CBL), standard GCPs cultures, S-cNS, and GF-cNS. We used three independent samples for each experimental condition. Transcripts expression was normalized on the mean expression level of four reference genes: Pgk1, Hprt, Gusb, Tfrc. Black dots: fold changes exceeding the maximum value of the given scale. Uncropped Western blot images related to this figure are displayed in Fig. S7.
