Fig. 4

Generation of DNMT1 knockout-rescue cell lines. a Examples of indel sequences for four single cell-derived clones. Schematic of the gRNA target site is shown above. PAM sequence is marked in orange. Cas9 cutting site is indicated by red arrow. Insertions are indicated by red letter. b Inhibition of exogenous DNMT1 expression in DNMT1-knockout cells caused cell death. The DNMT1knockout-rescue cells expressed GFP. Expression of GFP was inhibited by addition of Dox for 3 days. All cells died at day 7. c RT-qPCR analysis of the exogenous DNMT1 expression with or without Dox for a single cell-derived clone (n = 3, error bars showed mean ± SEM). d Western blot analysis of total DNMT1 expression with or without addition of Dox. WT cells were used as a control. e Quantification for the Western blot assay for Fig. 4d (n = 3, error bars showed mean ± SEM). f Luminometric Methylation Assay (LUMA) showed that the global DNA methylation levels were the same for the WT cells and DNMT1 knockout-rescue cells. Addition of Dox significantly decreased DNA methylation level in DNMT1 knockout-rescue cells (n = 3, error bars showed mean ± SEM)