Fig 2: JMJD6 expression is required for rDNA repeat integrity following DNA damage(A) Representative image showing individual NORs in a U2OS cell in metaphase stained using an anti UBF antibody. Scale bar 5 μm. (B) Ionizing radiation (2 Gy) exposure of U2OS cells, U2OS cells inactivated for JMJD6 expression (KO) and a clone from the latter cell line in which wild type JMJD6 was reintroduced (KO + wt). The number of UBF foci in cells was then counted and the results represented as box plot. For each point a minimum of 50 metaphases were scored. Results from one representative experiment from 2 independent experiments is shown. The p values of the difference between the indicated samples are shown (Wilcoxon test). (C) Western blot analysis of UBF expression in the different cell lines. (D) Evaluation of rDNA rearrangements by FISH combing. Representation of a rDNA repeat with the position of the DSB induced by AsiSI after OHTam treatment. The green and red lines represent the FISH probes used in DNA FISH combing experiments and targeting two adjacent sequences in the rDNA. An example of a canonical array (without rDNA rearrangement) is shown. Note that the green and red probes are in the same order throughout the array. An example of a non-canonical (with rDNA rearrangement indicated by a star) rDNA repeat is also shown. E. Quantification of non-canonical rearrangements measured before and after DSB induction in siRNA control and siRNA JMJD6-depleted cells. Quantification was performed on duplicate samples with more than 400 units examined on each samples. Results are the mean +/- s.e.m. of three independent experiments. * p<0.1 was considered as significant. p values of the difference in non induced DSB were calculated using Student t test and are p = 0.04 and p = 0.08 for siJMJD6-1 and siJMJD6-2, respectively. p values of the difference after DSB are p = 0.076 and p = 0.073 for siJMJD6-1 and siJMJD6-2, respectively.