Cells and cell culture
Human primary fibroblasts (IMR90 passage 12, ATCC) were cultured in EMEM supplemented with: 10% FBS (GIBCO, Life Technologies), 100 units/ml penicillin and 0,1 mg/ml streptomycin (GIBCO, Life Technologies), 1% NEAA (GIBCO, Life Technologies), the human colon cancer cell line HCT116  was cultured in DMEM supplemented with: 10% FBS, 100 units/ml penicillin and 0,1 mg/ml streptomycin. Cells were cultured in a humidified atmosphere of 4% CO2 in air at 37°C. For siRNAs transfection 1, 5 × 105 IMR90 cells and 2, 5 × 105 HCT116 cells were plated in 6-well dishes and incubated at 37°C. Specific siRNAs duplex were mixed with Lipofectamine2000 Reagent (Invitrogen, Life Technologies), according to manufacturer's recommendation and added to the cells. After 6 hours at 37°C, the transfection medium was replaced with fresh medium. To silence genes of interest post-transcriptionally, cells were transfected with siRNAs targeting DNMT1 (siDNMT1: 5'- AUU ACG UAA AGA AGA AUU A dTdT-3')  at final concentrations of 60, 80 and 100 nM, siRNAs targeting TP53 (si p53: 5'-GCA UGA ACC GGA GGC CCC AUtt-3')  and p14ARF (sip14: 5'-GAA GAU CAG GUC AUG AUG Att-3')  at a final concentration of 60 nM. All siRNAs were synthesized by Eurofins-MWG.
Real time RT-PCR
Primers to be used in Real time RT-PCR experiments were designed with Primer Express software (Applied Biosystems, Life Technologies) choosing amplicons of approximately 70-100 bp. The selected sequences were tested against public databases (BLAST) to confirm the identity of the genes. Total RNA was extracted from cells by using the RNAeasy mini kit (GE) or the "All prep DNA/RNA kit" (Qiagen) according to the manufacture's instruction. RNA was reverse-transcribed in a final volume of 100 μL using the High Capacity c-DNA Archive kit (Applied Biosystems, Life Technologies) for 10 minutes at 25°C and 2 hours at 37°C. For each sample 2 μL of cDNA, corresponding to approximately 100 nag of reverse transcribed RNA, was analyzed by Real time RT-PCR (95°C for 15 sec, 60°C for 60 sec repeated for 40 cycles), in quadruplicate, using the ABI PRISM 7300 instrument (Applied Biosystems). Real time RT-PCR was done in a final volume of 20 μl comprising 1x Master Mix SYBR Green (Applied Biosystems, Life Technologies) and 0, 3 μM of forward and reverse primers for: DNMT1 (Fw: 5'-GCACCTCATTTGCCGAATACA-3'; Rev: 5'-TCTCCTGCATCAGCCCAAATA-3'), TP53 (Fw: 5'-TTCGACATAGTGTGGTGGTGC-3'; Rev: 5'- AGTCAGAGCCAACCTCAGGC-3'), p21WAF1-Cip1 (Fw: 5'-CTGGAGACTCTCAGGGTCGA-3', Rev: 5'-CGGATTAGGGCTTCCTCTTG-3'), p14ARF (Fw: 5'- TGATGCTACTGAGGAGCCAGC-3', Rev: 5'-AGGGCCTTTCCTACCTGGCTC-3'), GAPDH (Fw: 5'-CTCATGACCACAGTCCATGCC-3', Rev: 5'-GCCATCCACAGTCTTCTGGGT-3'). Data were analyzed by averaging quadruplicates Ct (cycle threshold). Levels of RNA expression were determined by using the SDS software version (Applied Biosystems, Life Technologies) according to the 2-ΔΔct method. Levels of RNA expression of selected genes were normalized to the internal control GAPDH.
Protein concentration was measured using the Bio-Rad Protein Assay (Bio-Rad Laboratories). Proteins (40 μg) were separated by 10% SDS-PAGE containing 0,1% SDS and transferred to Hybond-C nitrocellulose membranes (Amersham Life Science) by electro-blotting. The membranes were sequentially incubated with goat anti-DNMT1, rabbit anti-p21WAF1-Cip1, rabbit anti-pRb (Santa Cruz, CA), mouse anti-p53 (Abcam, UK) as primary antibodies, and HRP-conjugated mouse, rabbit (Abcam, UK) and goat (Santa Cruz, CA) Gig as secondary antibodies. The target protein was detected with enhanced chemiluminescence Western blotting detection reagents (Pierce, Thermo Scientific). We used also mouse anti-β-tubulin (SIGMA, Italy) as internal control of loading. Membranes were stained by Ponca-Red to confirm equivalent loading of total protein in all lanes.
Cell cycle analysis
Asynchronously growing cells were treated with 80 nM siDNMT1 alone or together with 60 nM sip53 for 72 hours and released into complete medium with BromodeoxiUridine (BrdU, SIGMA, Italy) 0, 2 μg/ml for one hour. DNA content was determined using Propidium Iodide (PI, SIGMA, Italy) staining by treating cells with PBS solution containing 4 μg/ml of PI and 40 μg/ml RNase. Analysis of BrdU labelled cells was conducted as described previously  and samples were analyzed on a FACSCanto (Becton Dickinson). Analysis of IMR90-siDNMT1/p14ARF cell cycle was made by PI staining of DNA content. Experiments were repeated at least twice, 10000 events were analyzed by FACSDiva software.
Cells were seeded onto glass cover slides 24 hours before the analysis. They were then, treated with 0,2 μg/ml colcemid (Demecolcine, SIGMA, Italy) for three (HCT116) four (IMR90) or eight (IMR90-siDNMT1) hours. Cell were swollen in 75 mM KCl at 37°C, fixed with methanol/acetic acid (3:1 v/v) The cover slides were then air-dried and stained with 3% GIEMSA in phosphate-buffered saline for 10 minutes. Chromosome metaphases were evaluated using a Zeiss Axioskop microscope under a 63× objective. The analysis was repeated three times for each cell types.
Phosphorylated H2A.X immunostaining: IMR90 and HCT116 were grown on glass coverslips after siRNAs transfection. Cells on coverslips were fixed with cold methanol (-20°C), permeabilized with 0.01% Triton × (Sigma, Italy) and blocked with 0.1% BSA, both at room temperature. Then, coverslips were incubated with phosphorylated H2A.X rabbit polyclonal antibody (1:100 in PBS-BSA 0.1%, Upstate) overnight at 4°C, washed in PBS and incubated with a FITC-conjugated rabbit anti-mouse (Sigma, Italy) diluted 1:100 in PBS-BSA 0,1%) for 1 hour at 37°C. Nuclei were visualized with 1 μg/ml of 4',6-Diamidino-2-phenylindole (DAPI) and examined on a Zeiss Axioskop microscope (Zeiss, Germany) equipped for fluorescence under a 63× objective. Images were captured with a CCD digital camera (AxioCam, Zeiss).
Global DNA methylation analysis
"Methylamp Global DNA methylation quantification kit" (Epigentek, USA) was used to quantify global DNA methylation. 200 ng of genomic DNA extracted from cell samples was immobilized in the strip well specifically treated to have high affinity to DNA for 2 hours at 37°C. The methylated fraction of DNA can be recognized by sequential incubation with 5-methylcytosine antibody for 45 minutes at 37°C, and with secondary antibody for 60 minutes at room temperature. A colorimetric reaction allowed methylated DNA quantification at 450 nM with microplate reader. A methylated DNA was used as positive control. The analysis was repeated twice.
Slot Blot analysis
The DNA-slot blot analysis of 5-Methylcytosine was performed as described  with the following modifications. Purified DNA (100 μg/25 μL) from control, siDNMT1/p14ARF and siDNMT1/p53 IMR90 cells, was denatured at 100°C for 5 min and applied onto Hybond-N membrane (RPN203N, Amersham) using the Hybri-slot Manifold Apparatus (Whatman Biometra, Germany) under vacuum for 3 min. DNA was fixed to the membrane by exposing to UV light (90,000 μjoules/cm2) for 45 seconds using the Hoefer UVC 50 Crossliker (Amersham Biosciences). Samples were then treated with TBST, (0.05% Tween20), 5% w/v no-fat dry milk blocking buffer for 1 hour at RT and incubated with 5-Methylcytosine Monoclonal Antibody (1:500, cloneD33, Epigentek USA) in TBST (0.05% Tween20) 5% w/v no-fat dry milk blocking buffer for 1 hour at RT. The membrane was then washed with TBST (0.05% Tween20), 3 times for 10 minutes each, incubated with HRP conjugated secondary antibody anti-mouse (1:2000) (Abcam, UK) in blocking buffer for 1 hour at RT, washed again with TBST (0.05% Tween20), 3 times for 10 minutes each and once in double distilled water for 5 minutes, then developed by enhanced chemiluminescence detection reagents (Pierce Thermo Scientific) and image acquired with Chemidoc XSR Imaging System (BioRad). Spotted DNA quantity was detected by 0.02% w/v methylene blue in 0.3 M sodium acetate (pH5.2) followed by washing in double distilled water to reduce background noise. The relative 5 MeC Optical density (OD) value was calculated with Quantity One 4.6.7 software relative to DNA amount loaded and normalized to wild type sample.