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Reactions were terminated after 1 hour by adding loading buffer.

After cultivation for 24 hours, the cells were fixed for one hour by adding 4% paraformaldehyde and stained with Coomassie brilliant blue (0.2%) for 30 minutes at room temperature.

Subsequently, each well was washed twice with 200 μl of wash buffer (0.05% Tween 20 in PBS, pH 7.4) and blocked for 1 hour by adding 200 μl of PBS containing 2% BSA at 37°C.

For each immunoprecipitation, 300 μL of crosslinked sonicated sample was diluted with 1.2 ml ChIP buffer (1% triton X-100, 150 mM NaCl, 2 mM EDTA, 20 mM Tris-HCl pH 8.1 and protease inhibitors (Sigma Aldrich)) and precleared for one hour by adding 50 μL of protein A beads (50% slurry protein A-Sepharose, Amersham; 0.5 mg/mL lipid-free BSA, (Sigma Aldrich); and 0.2 mg/mL salmon sperm DNA in TE).

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Absorbance readings were taken at 0, 248 48, and 72 hours by adding WST-1 (Roche Diagnostics Division de Hoffman La Roche Limitée, Laval-des-Rapides, QC, Canada) as a cell proliferation reagent to each well (10% WST-1 in Dulbecco's Modified Eagle's Medium) followed by incubation at 37°C for 2 hours before reading at the indicated time points.

Fringe-GFP synthesis was induced for 3 hours by adding 1 mM CuSO4 in the medium followed by a 2-hour chase in the presence of 100 mg/ml cycloheximide.

500,000 cells were transfected and senescence was induced 24 hours after transfection for 6 hours by adding 20 nM 4-HT to the cell medium.

After 24 hours, cells were pulse-labeled for 3 hours by adding 0.5 μCi [H]thymidine/well (Amersham, Piscataway, NJ, USA).

Gene expression was induced at 30°C for 3 hours by adding 1.0 mmol/L Isopropyl-β-D-thio-galactoside (IPTG) when A600 reached to 0.8.

Slides were prepared for 4°C overnight incubation (12 hours) by adding a cocktail of MAP-Tau primary antibody (1 750) plus a wide-spectrum rabbit anti-cow cytokeratin antibody (Z0622; DAKO, Carpinteria, CA) diluted 1 100 in BSA/1X TBS-T.