Adaptation of mammalian cells to standard culture conditions results in 5hmC loss. (a) Dot-blot of genomic DNA from primary MEF tissue and cultured cells derived therefrom with a 5hmC-specific antibody (500 ng were loaded in all lanes). ssDNA, single-stranded DNA. (b) Quantification of global 5hmC levels by densitometric analysis of dot-blots (±SE). P, passage. (c) Quantification of total (5hmC + 5mC) modification levels in DNA by LUMA. DNA from triple knock-out (dnmt3a
-/-, dnmt3b-/-, dnmt1-/-) mouse ES cells (TKO) which contain <2% DNA methylation was used as a negative control and was set to a value of 0 and DNA methylation levels in MEF tissue were set to 100. Dnmt1
-/- MEF DNA (MEF DN ), which contains approximately 20% of normal methylation levels, is also shown (±SE). (d). Fluorescence microscopy images of MEFs during adaptation to culture stained with antibodies against 5mC (green) and 5hmC (red). DNA was counterstained with DAPI (blue). A merged 5mC/5hmC image is shown on the top right while two overexposed 5hmC-only images below show no nuclear staining after culture. (e) Bar-chart of quantitative RT-PCR showing reduced expression of Tet1 upon adaptation to culture. Values are the average of three biological replicates normalized to Gapdh expression ± standard deviation. *P < 0.05, Mann-Whitney U-test. (f) Dot-blot of DNA (1 μg) from male and female primary MEFs showing partial rescue of global 5hmC levels by addition of vitamin C (1,000 μM). Top row shows partial recovery of global 5hmC after addition of vitamin C at passage 2. Bottom row shows reduction of 5hmC loss after addition of vitamin C at passage 1. (g) Dot-blot of DNA (500 ng) from naïve CD4+ T cells and cultured cells derived therefrom with a 5hmC-specific antibody. Two replicate experiments are shown. (h) Quantification of global 5hmC levels by densitometric analysis of dot-blots shown in (g). Values were normalized to a methylene blue loading control.