Journal: Scientific Reports

Article Title: CX-5461 causes nucleolar compaction, alteration of peri- and intranucleolar chromatin arrangement, an increase in both heterochromatin and DNA damage response

doi: 10.1038/s41598-022-17923-4

Figure Lengend Snippet: Effect of CX-5461 exposure on HeLa cell cultures. ( a ) Reduced amount of newly synthesized rRNA (green) at 1 µM CX-5461 exposure for 1 h in comparison to control cells visualized by BrUTP-incorporation into nascent transcripts (representative images; widefield imaging); chart shows percentage of BrUTP-positives among a total of 220 cells in 1 experiment; ( b ) 1 h exposure shows significant reduction of cells undergoing DNA replication beginning from 500 nM CX-5461 onwards measured by EdU incorporation (mean 7600 cells per category, 3 independent experiments); statistics: bullet is always used as anchor point for comparison (read: first “ns” compares med_ctrl with solv_ctrl, second “ns” compares med_ctrl with 1 nM CX, etc.); ( c ) senescence is significantly induced at exposure to 1 µM CX-5461 for 1 h as measured by SA-ß-Gal staining (mean of 7200 DAPI-stained nuclei per category (8-bit grey values); 2 independent experiments); ( d ) viability shows no significant differences after 1 h CX-5461 incubation (1 nM–10 µM) measured with an MTT assay whereas incubation for 72 h ( e ) leads to significantly reduced viability, particularly from 500 nM CX-5461 onwards ( d , e : 3 independent experiments (triplicates)). Graph bars display means, error bars represent standard deviations; Student´s t -test was applied. Bar = 10 µm, ns not significant, *P < 0.05, **P < 0.01, ***P < 0.001.

Article Snippet: Stained coverslips of fixed cells were recorded with widefield epifluorescence microscope (Eclipse 800, Nikon; 40× (NA 1.0), 60× (NA 1.4) and 100× (NA 1.4)) or with an automated slide scanning system (VS120, Olympus; 40× (NA 1.25)); single confocal sections and stacks were acquired with the confocal microscope (above).

Techniques: Synthesized, Comparison, Control, Imaging, Staining, Incubation, MTT Assay

Journal: Scientific Reports

Article Title: CX-5461 causes nucleolar compaction, alteration of peri- and intranucleolar chromatin arrangement, an increase in both heterochromatin and DNA damage response

doi: 10.1038/s41598-022-17923-4

Figure Lengend Snippet: Changes of nucleolar morphology after CX-5461 exposure. ( a ) Time-lapse imaging for 1 h reveals significant nucleolar size reduction of HeLa cells expressing H2B-mCherry beginning after approx. 20 min after exposure to CX-5461 (timepoint 0 = 100% of nucleolar area (each 5 cells out of 1 experiment)). ( b ) DAPI staining shows changes of nucleolar morphology in fixed HeLa cells (representative images of several independent experiments; widefield imaging); note the distinct DAPI-positive ring after 1 µM CX-5461 treatment (blue arrows); note nucleolar dot (yellow arrowhead); ( c ) morphology factors roundness and circularity approach value 0.8 with increasing concentrations of CX-5461 (fixed HeLa cells); 1.0 is indicative of a perfect sphere; scatterplot for visual overview of the shape distributions (a mean of 282 nucleoli per category were evaluated from 3 independent experiments); ( d ) CX-5461 incubation does not show a significant change in the number of nucleoli per cell (counting of the number of cells containing 1, 2 or 3 and more nucleoli per cell; Hep3B cells; a mean of 64 cells with nucleoli per category were evaluated for 3 repeated measurements from 1 experiment); ( e ) the nucleolar size significantly decreases upon CX-5461 treatment (HeLa cells; a mean of 48 nucleoli per category were evaluated from 1 experiment); ( f ) frequently, DAPI-positive dots appear inside nucleoli after 1 µM CX-5461 exposure for one hour in fixed cells (yellow arrows; single confocal section); 3-D reconstruction of nucleolar interior (grey) and perinucleolar chromatin (skin-colour) reveals tunnel-like nature of chromatin strands running through nucleoli (representative HeLa cells with tunnels). ( g ) significant increase of nucleolar surface (perimeter) coverage by the bright DAPI positive perinucleolar heterochromatin upon CX-5461-treatment (fixed HeLa cells; a mean of 45 nucleoli per category evaluated from 2 independent experiments); ( h ) likewise, time-lapse imaging for 1 h revealed significant increase in perinucleolar heterochromatin upon CX-5461 treatment evaluated by the bright perinucleolar signal in H2B-YFP expressing cells; significant differences can be observed after approximately 10 min (each 9 cells out of 1 experiment); bar and line graph display means, error bars represent standard deviations; box plot charts show first and third quartile, sample median and whiskers (1.5× interquartile range); Student´s t -test was applied. Bars = 1 µm ( f ), 10 µm ( a ); ns not significant, *P < 0.05, **P < 0.01, ***P < 0.001.

Article Snippet: Stained coverslips of fixed cells were recorded with widefield epifluorescence microscope (Eclipse 800, Nikon; 40× (NA 1.0), 60× (NA 1.4) and 100× (NA 1.4)) or with an automated slide scanning system (VS120, Olympus; 40× (NA 1.25)); single confocal sections and stacks were acquired with the confocal microscope (above).

Techniques: Imaging, Expressing, Staining, Incubation, Cell Counting

Journal: Scientific Reports

Article Title: CX-5461 causes nucleolar compaction, alteration of peri- and intranucleolar chromatin arrangement, an increase in both heterochromatin and DNA damage response

doi: 10.1038/s41598-022-17923-4

Figure Lengend Snippet: Distribution of γH2AX and topo I after CX-5461 treatment; fixed HeLa cells. ( a ) Representative images of antibody labelling; overlays of IF (green) and DAPI staining (red; false colour); ( b ) γH2AX and topo I show high label denisties over nucleoli and perinucleolar areas after CX-5461 exposure (widefield imaging; 20 randomly chosen nuclei were evaluated in 2 independent experiments each); Graph bars display means, error bars represent standard deviations of mean, background-corrected grey values; Student´s t -test was applied. Bars = 10 µm; ns not significant, *P < 0.05, **P < 0.01, ***P < 0.001.

Article Snippet: Stained coverslips of fixed cells were recorded with widefield epifluorescence microscope (Eclipse 800, Nikon; 40× (NA 1.0), 60× (NA 1.4) and 100× (NA 1.4)) or with an automated slide scanning system (VS120, Olympus; 40× (NA 1.25)); single confocal sections and stacks were acquired with the confocal microscope (above).

Techniques: Staining, Imaging