Figure 3: DAXX disruption compromises gene expression and restores H3.3 deposition in PTEN-deficient cells.

(a,b) Total proteins were analysed by western blot in PTEN-deficient (a) or PTEN-expressing (b) GBM-spheres transduced with lentivirus shRNA control (shLuc) or shRNAs targeting DAXX (shDaxx). Actin is a loading control. (c,d) anti-H3.3 ChIP-qPCR was performed in PTEN-deficient (c) or PTEN-WT (d) GBM-spheres with stable knockdown of DAXX or with shControl. (e) ChIP-qPCR of H3.3 in Daxx-kd/PTEN-deficient GBM-spheres expressing a HA-DAXX-shRNA-resistant vector or PTEN-WT. For ChIP assays, bar graphs indicate fold enrichment of H3.3 over input (n=3 biological samples with three replicates each, **P<0.001, ***P<0.0001, one-way ANOVA). (f) Immunoblot analysis of HA-DAXX and PTEN expression in DAXX-kd or shControl GSC23 neurospheres. NS: no significant differences. Error bars represent s.e.m. from three different experiments. Numbers under the blots indicate fold ratios of protein levels relative to shControl after normalization to actin.