Sucrose

The following microbiological media components were used: Bacto malt extract was purchased from Becton Dickinson (Környe, Hungary) and yeast extract, NaCl and sucrose were purchased from Molar Chemicals (Halásztelek, Hungary). All solvents used for extraction were purchased from Molar Chemicals Hungary and were of analytical grade. The solvents used for the various solvent systems and HPLC-UV measurements were of gradient grade or super gradient grade and were purchased from VWR International (Debrecen, Hungary). The AF standard mixture (including AFB₁, AFG₁, AFB₂ and AFG₂) was purchased from Sigma-Aldrich (Budapest, Hungary).

U2OS cells were washed with PBS (Phosphate-buffered saline), then rinsed twice for 3 minutes with CSK (Cytoskeletal) buffer [10 mM HEPES pH 7.0 (Sigma-Aldrich), 100 mM NaCl (Sigma-Aldrich), 300 mM sucrose (Molar Chemicals), 3 mM MgCl₂ (Molar Chemicals), 0.7% Triton-X-100 (Fluka) and 0.3 mg/ml RNase A (Sigma-Aldrich)]. Cells were rinsed twice with PBS, then fixed with 4% formaldehyde (Sigma-Aldrich) for 10 minutes. After washing, cells were permeabilised for 5 minutes in PBS containing 0.2% Triton-X-100 (Fluka). Non-specific staining was blocked with 5% BSA in PBST [0.1% Tween 20 (Molar Chemicals) in PBS] for 20 minutes. Cells were incubated with the following primary antibodies: anti-p53 (Dako, IS616), N-20 anti-RPB1 (Santa Cruz, sc899), anti-S2P RPB1 (Abcam, ab5095), anti-S5P RPB1 (Abcam, ab5131), anti-S139P H2AX (Millipore, 05-636-I) and anti-S139P H2AX (ab2893). After washing, the following secondary antibodies were used: GAM Alexa 488 (Molecular Probes, A11029) and GAR DyLight 550 (Abcam, ab96984). Both the primary and secondary antibodies were applied in 1% BSA-PBST. Coverslips were mounted on glass slides using DAPI containing ProLong Gold Antifade reagent (Life Technologies). Samples were visualised with Olympus FluoView FV1000 confocal microscope. The same exposition time was used to capture each image.