Fig. 4

Targeted Casp8AP2 methylation increases drug resistance. a Two-component system for visualizing targeted DNA methylation. Components were co-transfected into the same cells, and stable clones were selected by adding neomycin and hygromycin to the culture medium. Detection of targeted DNA methylation is described in the “Methods”. b Detection of targeted DNA methylation. Upstream and downstream target regions are indicated by the regions between two arrows in the upper panel. Short vertical bars indicate CpG sites. Targeted DNA methylation was detected by qMSP with primer pairs (indicated by arrowheads in the upper panel), and mock-treated cells were used as a control (middle panel). Expression of the EGFP reporter gene was detected by ELISA (lower panel). c Increased Casp8AP2 methylation (upper panel) and decreased Casp8AP2 expression (lower panel) after targeted DNA methylation in MSCs (left) and MDA-MB-231 breast cancer cells (right). Untreated cells are designated as control (ctrl), and cells treated with transfection reagent only are designated as (−). d Increased cell survival after targeted Casp8AP2 methylation. MDA-MB-231 cells were transfected with in vitro methylated (S.ssI) or unmethylated Casp8AP2 DNA and then challenged with different concentrations of cisplatin (upper panel) and taxol (lower panel). Cell survival after drug treatment was detected by MTT assay