DNA methylation is the most studied epigenetic modification with a wide range of regulatory functions in mammalian genomes. It almost exclusively resides on CpG dinucleotides and, among others, plays important roles in early embryo development, onset, and maintenance of cancer. During the past 3 decades, many approaches have been developed to discriminate methylated from unmethylated DNA including antibody-based enrichment of methylated DNA, restriction enzyme-based, or hybridization-based methods. The conversion of unmethylated cytosines to uracils by sodium or ammonium bisulfite is regarded as golden standard as this approach requires no enzymatic reaction and provides deep and reliable insight in methylation patterns at single-base resolution. Nowadays, there are many commercial kits for bisulfite conversion available but they perform differently and also vary in protocols and chemicals used. Here, we provide the first comprehensive and comparative evaluation of bisulfite conversion kits observing major differences in conversion efficiency and DNA degradation which greatly affect the performance of downstream applications, ie, polymerase chain reactions (PCRs). Moreover, deep sequencing of amplicons containing oxidized derivates of 5ʹ-methylC shows that none of the tested kits efficiently converts 5ʹ-formylC without substantial conversion of 5ʹ-methylC or 5ʹ-hydroxymethylC. Consequently, we developed a robust and easy-to-use protocol that allows maximal discrimination between 5ʹ-formylC and 5ʹ-methylC/5ʹ-hydroxymethylC with low DNA degradation and high PCR efficiency on the bisulfite-treated DNA. We highly recommend to use our time- and cost-efficient protocol for any genome-wide or local high-resolution bisulfite sequencing application to minimize conversion-dependent error rates.

Nerve/glial antigen (NG)2 expression crucially determines the aggressiveness of glioblastoma multiforme (GBM). Recent evidence suggests that protein kinase CK2 regulates NG2 expression. Therefore, we investigated in the present study whether CK2 inhibition suppresses proliferation and migration of NG2-positive GBM cells. For this purpose, CK2 activity was suppressed in the NG2-positive cell lines A1207 and U87 by the pharmacological inhibitor CX-4945 and CRISPR/Cas9-mediated knockout of CK2α. As shown by quantitative real-time PCR, luciferase-reporter assays, flow cytometry and western blot, this significantly reduced NG2 gene and protein expression when compared to vehicle-treated and wild type controls. In addition, CK2 inhibition markedly reduced NG2-dependent A1207 and U87 cell proliferation and migration. The Cancer Genome Atlas (TCGA)-based data further revealed not only a high expression of both NG2 and CK2 in GBM but also a positive correlation between the mRNA expression of the two proteins. Finally, we verified a decreased NG2 expression after CX-4945 treatment in patient-derived GBM cells. These findings indicate that the inhibition of CK2 represents a promising approach to suppress the aggressive molecular signature of NG2-positive GBM cells. Therefore, CX-4945 may be a suitable drug for the future treatment of NG2-positive GBM.