Figures and data
PNAs formation after diverse stress-inducing stimuli and topoisomerase downregulation.
(A) Structural types of PML nucleolar associations (PNAs) – ‘bowls’, ‘funnels’, ‘balloons’, and PML nucleolus-derived structures (PML-NDS) occurring in RPE-1hTERTcells after treatment with 0.75 μM doxorubicin detected by indirect immunofluorescence with anti-PML antibody (green) and counter-stained with TOTO-3 (red, for nucleoli visualization) and DAPI (blue). Scale bar, 10 μm. (B) Quantification of the percentage of RPE-1hTERTcells containing PNAs, 48 hours after treatment with various stress-inducing stimuli. The stress stimuli were divided into five categories according to their mechanism of action: 1) inhibitors of topoisomerases, 2) inhibitors of RNAP I, 3) inhibitors of pre-RNA processing, 4) inductors of replication stress, and 5) other stressors. p53 stabilization, γH2AX foci formation, PAF49 segregation, and TOP1 or TOP2a decline have been assessed for each treatment. (C) The pattern of PML (green) and B23 (red) in RPE-1hTERTcells, visualized by indirect immunofluorescence, after transfection with esiRNAs targeting topoisomerases 1 and 2alpha, with siRNA targeting topoisomerase 2beta, or with non-targeting siRNA, respectively. Scale bar, 10 μm. (D) RPE-1hTERT cells were pre-treated with 10 nM AMD for 5 hours or with 5 μM CX-5461 for 2 hours to inhibit RNAPI. The cells were then treated with 0.375 μM or 0.75 μM doxorubicin or transfected with esiRNA targeting TOP1 for 48 hours. The bar graphs show the percentage of cells containing either PML-NDS or bowls/funnels/balloons for three independent experiments (graph 1), for a single experiment (graph 2), or for three biological replicates (graph 3). Results are presented as a mean ± s.d.
Inhibition of DNA repair augmented the PNAs formation.
RPE-1hTERT cells were treated with doxorubicin and three concentrations of B02 or with etoposide after downregulation of TDP2 by RNA interference. After the treatment, the PML (green) and nucleolar marker B23 (red) were visualized by indirect immunofluorescence and wide-field fluorescent microscopy, and a number of nuclei with PNAS were analyzed. (A) The bar graph represents the percentage of nuclei with PNAs after 2-day-long treatment with doxorubicin (0.375 µM or 0.56 µM), three concentrations of B02 (5, 10, and 20 µM), and corresponding concentrations of DMSO as a mock. (B) The bar graph represents the distribution of individual types of PNAs after the same treatments as in shown in (A). (C) Representative cells after 2-day-long treatment with 0.375 µM doxorubicin combined with 20 µM B02 or 0.1% DMSO (mock). (D) Representative cells after 2-day-long treatment with 0.56 µM doxorubicin combined with 20 µM B02 or 0.1% DMSO (mock). (E) The bar graph represents the percentage of nuclei with PNAs after 4 days of recovery from 2-day-long treatments with doxorubicin (0.375 µM or 0.56 µM) together with three concentrations of B02 (5, 10, and 20 µM), and corresponding concentrations of DMSO. (F) Representative cells after 4 days of recovery from 2-day-long treatment with 0.375 µM doxorubicin combined with 20 µM B02 or 0.1% DMSO (mock). (G) Representative cells after 4 days of recovery from 2-day-long treatment with 0.56 µM doxorubicin combined with 20 µM B02 or 0.1% DMSO (mock). (H) The bar graph represents the percentage of nuclei with PNAs after 2-day-long treatment with 5 µM etoposide in cells where TDP2 was downregulated by RNA interference. In all experiments, at least three biological replicates were evaluated. Results are presented as a mean ± s.d. Student’s t-test was used for statistical evaluation. Asterisks indicate the following: ****P < 0.0001, ***P < 0.001, **P < 0.01, *P < 0.05. Scale bar, 20 μm.
PNAs encircle rDNA and DJ loci containing DNA DSB after doxorubicin treatment.
RPE-1hTERT cells were treated with 0.75 µM doxorubicin for 2 days and recovered from the treatment for one and four days. The proliferating cells were used as a control. The localization of rDNA, DJ, PML, and 53BP1 was analyzed using immuno-FISH staining and confocal microscopy. (A) The scheme of a human acrocentric chromosome. The position of probes used for the detection of the rDNA locus (blue) and DJ locus (grey) is shown. (B) The representative nuclei and the nucleoli with and without PNAs in control and treated cells are shown. rDNA (red), DJ (white), PML (green), and 53BP1 (red). (C) The extent of PML-rDNA and PML-DJ size-based colocalization calculated for individual nucleoli of treated and untreated cells with respect to the presence of PNAs is shown as a scatter plot. The median with an interquartile range is shown. The colocalization was calculated using Fiji(ImageJ)/ Mosaic/Segmentation/Squassh plugin. The number of analyzed nucleoli in each group was: ctrl (n=26); 2 days + PNAs (n=28); 2 days without PNAs (n=18); 1-day-long recovery + PNAs (n=38); 1-day-long recovery without PNAs (n=20); 4-day-long recovery + PNAs (n=23); 4-day-long recovery without PNAs (n=24). (D) The extent of 53BP1-rDNA and 53BP1-DJ size-based colocalization calculated for individual nucleoli of treated and untreated cells with respect to the presence of PNAs is shown as a scatter plot. The median with interquartile range is shown. The same collection of nucleoli was used as presented in (C). (E) The example of analysis that was used to identify whether rDNA/DJ with DNA DSB colocalized with PNAs. The images of the representative nucleoli (2 days doxorubicin, 1- and 4-days recovery) after deconvolution, segmentation, and its 3D model are shown. Note, that the signal of all observed markers was identified as a unique 3D object. rDNA (blue), DJ (white), PML (green), and 53BP1 (red). (F) The combined bar graph shows the percentage of PNAs containing rDNA/DJ with DNA DSB, rDNA/DJ without DNA DSB, and PNAs in which the rDNA/DJ signal was not detected. The number of analyzed nucleoli: 2 days doxorubicin (n=28), 1-day-long recovery (n=39), 4-day-long recovery (n=21). Student’s t-test was used for statistical evaluation. Asterisks indicate the following: ****P < 0.0001, ***P < 0.001, **P < 0.01, *P < 0.05. Scale bars, 10 μm (nuclei in B and E) and 5 μm (nucleoli in B).
The DNA damage introduced into the rDNA locus by endonuclease I-PpoI induces PML-NDS.
(A) Scheme of inducible expression of endonuclease I-PpoI in RPE-1hTERT cells and the position of I-PpoI cleavage site in rDNA locus. (B) Scheme of the experimental setup used in all experiments presented. Briefly, I-PpoI was activated by doxycycline and Shield for 24 hours, then the medium was exchanged, and the cells were analyzed upon the recovery phase (0, 1, 2, and 5 days). (C) The representative images obtained by indirect immunofluorescence and confocal microscopy (Stellaris) show the localization of 53BP1 (a marker of DNA DSB, red), and PML (green) upon 1-day-long activation of I-PpoI and during recovery from I-PpoI insult. The nucleus and nucleolus were marked by DAPI (blue), and TOTO-3 (white), respectively. Only one layer from the several sections is presented. (D) The level of DNA DSB upon the recovery from I-PpoI insult was obtained by detecting the 53BP1 (a marker of DNA DSB) by indirect immunofluorescence and high-content microscopy unit (ScanR). The histogram represents the frequency of nuclei (%) with the same number of 53BP1 foci. The bin center used for analysis was 2. (E) The number of nuclei with PNAs upon the recovery from I-PpoI insult was obtained by detection of PML using indirect immunofluorescence and a high-content microscopy unit (ScanR). The quantification was done manually by the evaluation of PML localization in more than 200 nuclei. The bar graph represents the percentage of nuclei with PNAs. Results are presented as a mean ± s.d. obtained from three biological replicates. (F) The representative images obtained by indirect immunofluorescence and confocal microscopy (Stellaris) show the correlation between the localization of B23 (red) in PML-NDS (PML, green) upon the recovery from I-PpoI insult. The nucleus and nucleolus were marked by DAPI (blue), and TOTO-3 (white), respectively. Only one layer from the several sections is presented. (G) The representative images obtained by indirect immunofluorescence and wide-field microscopy show the correlation between the localization of DHX9 (red) and PML-NDS (PML, green) upon the recovery from I-PpoI insult. The nucleus and nucleolus were marked by DAPI (blue), and TOTO-3 (white), respectively. (H-I) The representative images obtained by indirect immunofluorescence and confocal microscopy (Stellaris) show the localization of UBF ((H), a marker of rDNA; red) or PAF49((I), a subunit of RNAPI; red) in PML-NDS (PML, green) upon the recovery from I-PpoI insult. One layer of the nucleus and three sequential layers of nucleolus with PML-NDS are presented. The nucleus and nucleolus were marked by DAPI (blue) and by TOTO-3 (white), respectively. Scale bars, 10 μm (nuclei C, F, G, H, and I) and 5 μm (nucleoli H and I).
Inhibition of RAD51/homologous recombination suppressed the formation of I-PpoI-induced PML-NDS.
(A) Scheme of the experimental setup used in all experiments presented in this figure. Briefly, I-PpoI was activated in RPE-1hTERT-I-PpoI cell clones 1A11 and 1H4 by doxycycline and Shield for 24 hours. The inhibitors of DNA PK (1 µM Nu7441) or RAD51 (10 µM B02) were applied individually or both together. After the 24-hour long activation, the medium was exchanged and the cells were analyzed during the recovery phase (0, 1, 2, and 5 days). (B and C) During the recovery phase the level of DNA DSB was quantified by the detection of 53BP1 (a marker DNA DSB) using indirect immunofluorescence and a high-content microscopy unit (ScanR). The data from three independent biological replicates were pooled together and represented as the Whiskers box (10 – 90 percentile) or as the histograms showing the frequency of nuclei (%) with the same number of 53BP1 foci. The bin center used for analysis was 2. Clones 1A11 and 1H4 are shown in (B) and (C), respectively. (D and E) The number of nuclei with PNAs upon the recovery from I-PpoI insult and simultaneous inhibition of particular DNA damage repair pathway was obtained by detection of PML using indirect immunofluorescence and a high-content microscopy unit (ScanR). The quantification was done manually by the evaluation of PML localization in more than 200 nuclei. The bar plot represents the fold-change of the percentage of nuclei with PNAs relatively to mock. Results are presented as a mean ± s.d. obtained from three biological replicates. Asterisks indicate the following: ****P < 0.0001, ***P < 0.001, **P < 0.01, *P < 0.05.
