Fig. 3.

Validation of melanocyte and fibroblast cell systems for experimental depletion of DNMT1. a The DNMT1 mRNA expression levels were measured by quantitative RT-PCR in HNEM-hTERT- and BJ-hTERT-derived pTctrl and pTshDNMT1 clones. The clones were exposed (+ doxy) or not (− doxy) to doxycycline (1 μg/mL—7 days). Expression values represent the mean (± sem) of at least three independent experiments. ****p < 0.0001. b Protein levels of DNMT1 and pKu80 (a loading control) were evaluated by Western-blot in the different clones, either with or without exposure to doxycycline. c The number of population doublings of HNEM-hTERT- and BJ-hTERT-derived clones, with or without 7 and 14 days doxycycline exposure, were determined by cell counting. Results derive from two independent experiments. d FACS analysis was performed on HNEM-hTERT- and BJ-hTERT-derived pT-shDNMT1 clones exposed or not to doxycycline (7 days). Propidium iodide staining (X axis) was measured and the percentage of cells in each cell cycle phase was determined