Computational protocol: Heterozygous mutations in PALB2 cause DNA replication and damage response defects

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Protocol publication

[…] Exponential growing cells were pulse labelled with 25 μM CldU (Sigma) followed by 250 μM IdU (Sigma) for the indicated times. For fibre analysis, after damage induction CldU and IdU media was given for indicated time periods and 2 mM HU or 10 μM ET were performed during the last 30 min of the CldU pulse. For analysis of Cdk-dependence of origin firing, roscovitine (Sigma) was added to give a final concentration of 50 or 100 μM 1 h before the addition of CldU. Labelled cells were harvested and DNA fibre spreads prepared on microscope glass slides by addition of 200 mM Tris-HCl pH 7.4, 50 mM EDTA, 0.5% SDS. Acid-treated fibre spreads were stained with monoclonal rat anti-BrdU (AbD Serotec, 1:1,000) followed by monoclonal mouse anti-BrdU (Becton Dickinson, 1:1,500). Secondary antibodies were goat anti-rat Alexa Fluor 555 and goat anti-mouse Alexa Fluor 488 (both Molecular Probes, 1:500). Fibres were examined using fluorescence microscope Axioplan 2 (Zeiss, Germany). CldU and IdU tracks were measured using ImageJ and micrometre values were converted into kilobases. At least 200 replication forks were analysed. Different classes of tracks were classified; red-green (ongoing replication), red (stalled or terminated forks), green (2nd pulse origin) and red-green-green-red (1st pulse origin). Frequencies were calculated using ImageJ software. [...] Independent samples Student’s t-test for normally distributed variables and Mann–Whitney U-test for non-normally distributed variables was used to determine the statistical difference in ATR and PALB2 expression, origin firing, Chk1 and Chk2 phosphorylation and in the number of different chromosomal aberrations between the two groups categorized according to their PALB2 c.1592delT mutation status. Correlation between data sets was evaluated using Spearman’s rank coefficient. Comparisons were done with IBM SPSS Statistics software (version 20.0, IBM Corp) and all P-values were two-sided. Dot plots were used to illustrate the distribution of the responses. […]

Pipeline specifications

Software tools ImageJ, SPSS
Applications Miscellaneous, Microscopic phenotype analysis
Diseases Breast Neoplasms, Neoplasms, Neoplastic Syndromes, Hereditary