Figure 6: Jmjd3 is required for Shh-subtype medulloblastoma cell growth.

(a) H&E staining of sections of a P90 normal cerebellum and one with a SmoM2-induced MB. (b) Levels of histone modifications and EZH2 in normal cerebellum (CB) and SmoM2-induced medulloblastoma (MB) were analysed by immunostaining. IGL=internal granule layers. (c) Western blots indicating global levels of histone modifications and EZH2 in normal CB and SmoM2 MB. (d) RT–qPCR analysis of EZH2 in NIH3T3 and MEF cells and in CB and MB samples (means±s.d., n=3). (e) ChIP-qPCR analysis of histone modification levels in the Gli1 regulatory region in MB and normal CB. Histone H3 ChIP was used for ChIP normalization. (means±s.d., n=3). (f) Cultured SmoM2 medulloblastoma tumour cells were infected with lentiviruses expressing either Jmjd3 shRNA or scrambled control. Levels of Shh target genes and medulloblastoma progenitor marker Math1 were measured with RT–qPCR (means±s.d., n=3). (g) The H3K27me3 level in the Gli1 gene regulatory region was increased after inhibition of Jmjd3 expression (means±s.d., n=3). (h) Cultured SmoM2 tumour cells were infected with viruses expressing control or shRNA targeting Jmjd3 or Gli1/2. The survival rates of cells relative to the control culture were measured using the ATP cell viability assay (means±s.d., n=3). Significance was determined by Student’s t-test or ANOVA with post hoc t-test; ** indicates P<0.01 and * indicates P<0.05.