Análisis de resultados

The histone variant H2AX is phosphorylated at serine 139 in response to DNA double-strand breaks, leading to the formation of γ-H2AX foci at the damaged sites. Phosphorylation is mediated

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by activation of the phosphatidylinositol-3-OH-kinase-like family of protein kinases, DNA-PKcs, ATM and ATR, and is used as a connection point for accumulation of central components of the signal cascade initiated by the DNA damage (Rogakou et al. 1998). DNA double-strand breaks were quantified by immuncytochemically staining NB cells for γ-H2AX foci and counting them in a total number of 100 cells per sample. Cells were grown in complete medium on sterile coverslips in a 24-well plate. Transfection with siRNA against YB-1 was performed at 70 % confluency as described above. Medium was changed 24 h after transfection. Cells were then left untreated or irradiated with gamma radiation at 1 Grey 48 h after transfection, depending on the experiment. For γ-H2AX staining, cells were washed with PBS, fixed on coverslips with 4 % parafomaldehyde for 10 min at room temperature, then washed three times with PBS and either stored at 4 °C or processed immediately. All incubations were conducted at room temperature unless otherwise stated. Cells were permeabilized in PBS containing 0.2 % Tritron X-100 and 1 % BSA for 10 min on ice, then washed again with PBS containing 1 % BSA for 2 x 5 min and incubated in blocking solution (PBS containing 3 % BSA and 0.2 M glycine) for 1 h. A polyclonal antibody against phospho-H2AX (Ser 139, Millipore) was diluted 1:200 in PBS-T-05 containing 1 % BSA for 1 h. Unspecific antibody binding was removed by a 1 h washing step in PBS-T-05 containing 1 % BSA. Alexa Flour 594-labeled Molecular Probes® anti-mouse IgG (Invitrogen) was diluted 1:2000 in PBS containing 0.5 % goat serum and incubated with the cells for 1 h. Cells were washed twice in PBS, then nuclei were counterstained with a 250 ng/ml DAPI (H33342). After washing another time with PBS, cells were mounted onto microscope slides using Vectashield® Flourescent Mounting Medium (Vector Laboratories) and stored at 4 °C until observation.

3.15.

I

Cells were grown in complete medium on sterile coverslips in 24-well plates for YB-1 immunocytochemistry. When cultures reached 60 % confluency, cells were washed with PBS, fixed on coverslips with 4 % parafomaldehyde for 10 min at room temperature, then washed three times with PBS and either stored at 4 °C or processed immediately. All incubations were conducted at room temperature unless otherwise stated. Cells were permeabilized in PBS containing 0.2 % Tritron X-100 and 1 % BSA for 10 min on ice then washed twice for 5 min with PBS containing 1 % BSA. A demasking step was performed next to allow better binding of the antibody to intracellular proteins. The coverslips were incubated 5 min in saline-sodium citrate (SSC) buffer in a steam bath, then slowly cooled to room temperature 3 times in succession using fresh SSC buffer each time. To prevent unspecific binding, the coverslips were then incubated in blocking solution for 1 h. The primary antibody targeting YB-1 (Abcam) was diluted 1:100 in PBS-T-05 containing 1 % BSA, and cells were incubated for 1 h. Unspecific antibody binding was removed by a 1 h

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washing step in PBS-T-05 containing 1 % BSA before incubation with the secondary antibody, donkey anti-rabbit IgG labeled with Texas Red (Dako), diluted 1:200 in PBS for 1 h. Cells were washed twice in PBS, then nuclei were counterstained in a 250 ng/ml solution of DAPI (H33342) in PBS before washing again once with PBS. Coverslips were mounted onto microscope slides using Vectashield® Flourescent Mounting Medium (Vector Laboratories) and stored at 4 °C.

20 x Saline-sodium citrate (SSC) buffer

NaCl 175.3 g

Na3Citrate x 2H2O 88.2 g

ddH2O to 1 l

3.16.

A

A

SK-N-BE and SK-N-BEcp cells were transfected with siRNA targeting YB-1, then pulse-treated with cisplatin for 4 h (SK-N-BE cells with 12.5 µg/ml and SK-N-BEcp with 17.5 µg/ml) 48 h after transfection, before transferring to fresh medium without cisplatin and an additional 24 h of culture. After trypsinization and washing, cells were pelleted by centrifugation, resuspended in PBS containing 25% starch solution and spotted onto precoated microscopic slides. The following steps were performed in the Institute for Cell Biology, University Hospital Essen (Thomale Group). After air drying the slides, cells were fixed in methanol at −20°C for 12 h and then rehydrated in PBS for 10 min at 25° C. Cellular RNA was digested by RNase treatment (RNase A, 200 µg/ml; RNase T1, 50 U/ml in PBS; 100 µl per slide) for 1 h at 37°C in a humidified chamber. Each of the steps was followed by washing with PBS. Nuclear DNA was partly denatured by mild treatment with alkali (70 mM NaOH, 140 mM NaCl, 40% methanol v/v; 5 min at 0° C) and cellular proteins were removed by a two-step proteolytic procedure: Samples were first digested with prewarmed pepsin (Roche; 200 µg/ml;) for 5 min at 37°C, and then with prewarmed proteinase K (2 µg/ml in 20 mM Tris–HCl, 2 mM CaCl2, pH 7.5) for 5 min at 37°C. After blocking with casein (1% in PBS)

for 30 min at 25°C, slides were incubated with anti-Pt-DNA antibodies (in-house antibody, 0.1 µg/ml in PBS containing 1% casein and 200 µg of sonicated calf thymus DNA/ml; 2 h; 37°C). After washing with 0.05% Tween 20 in PBS for 2 min at 25°C, three-step sandwich immunostaining was performed by subsequent incubation with: (i) FITC-labeled goat anti-rat Ig (Dianova, Hamburg, Germany; 6.5 µg/ml in PBS, 1% casein; 45 min; 37°C), (ii) ALEXA FLUOR 488-labeled rabbit anti-FITC (5 µg/ml in PBS, 1% casein; 45 min at 37°C; Molecular Probes) and (iii) ALEXA FLUOR 488 goat anti-rabbit IgG (5 µg/ml in PBS, 1% casein; 45 min, 37°C; Molecular Probes). Nuclear DNA was counterstained with DAPI (1µg/ml in PBS) for 30 min at 25°C, and cells were mounted in Vectashield mounting medium. Position, area and intensities of

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signals from individual cell nuclei were visualized by fluorescence microscopy, and recorded using an image analysis system. Antibody-derived signals were normalized for the actual DNA content of a given cell and expressed as arbitrary fluorescence units (AFU).