Figure 3.

DCA induces PKM2–Oct4 interaction and represses Oct4 transcriptional activity. (a) Expression of Oct4 protein in NSCs and glioma spheroids grown as spheroids treated or not with DCA. The protein expression normalized to the expression of actin as indicated in the image as the mean±S.D. from three independent experiments. (b) PKM2 was immunoprecipitated from NSCs or glioma spheroids (P7^sph) as described in experimental procedures. The amount of co-immunoprecipitated Oct4 was revealed by immunoblot, quantified and normalized by PKM2 expression (**P<0.01). (c) PKM2–Oct4 complexes (red dots) were visualized by proximity-ligation assay in control or DCA-treated glioma spheroids. Nuclei were stained in blue by DAPI. (d) The mRNA expression of four target genes of Oct4 was measured in DCA-treated NSCs or glioma spheroids (P7^sph) and normalized by their expression in the corresponding control cells (*P<0.05; **P<0.01). (e) Chromatin was extracted from NSCs or glioma spheroids. The amount of Oct4 present on the chromatin was revealed by immunoblot, quantified and normalized by the ratio Oct4/H3 calculated (**P<0.01)