Fig. 3.
The mechanical properties of the nucleus are not altered by transcription inhibition. (A,B) Example images (A) and graphs (B) of the relative immunofluorescence signal of a euchromatin marker (H3K9ac, orange) and a heterochromatin marker (H3K9me2,3, gray) in untreated (‘unt’), VPA-treated, VPA- and α-amanitin-treated (‘VPA+aam’), and α-amanitin-treated (‘aam’) cells. The means of three biological replicates with n=100–300 cells each are shown as dots. (C) Example images of side and top-down views of Hoechst-labeled nuclei captured using spinning-disk confocal microscopy. There is no yellow dotted line. (D) Example line scans through the side-on image of the nucleus of the VPA-treated cell from panel C, from which the full-width at half maximum (FWHM) values from two different line scans were averaged to determine the height of each nucleus. (E) Individual (dots) and average (bar) measurements of nuclear height for each condition (n=15 nuclei for each condition). (F) Left: example images of a micromanipulation force-extension measurement, with an isolated nucleus from MEF V−/− cells pulled by the ‘pull’ pipette (bottom right) to extend the nucleus and held by the ‘force’ pipette (top left), the deflection of which multiplied by a precalibrated spring constant measures force. Right: example force-extension graph of control (‘unt’) and chromatin-decompacted (‘VPA’) cells over short and long regimes. The dashed line indicates the crossover from the short-extension to the long-extension regime. (G) Graph of the individual (dots) and average (bar) short-extension nuclear spring constants (‘unt’, n=21; ‘VPA’, n=15; ‘VPA+aam’, n=12; ‘aam’, n=12). Long-extension spring constants did not change for all conditions (P>0.05, Fig. S3B). α-Amanitin treatment was for 24 h. Error bars represent standard error. ns, not significant, P>0.05; *P<0.05; **P<0.01 (two-tailed unpaired Student's t-test). a.u., arbitrary units. Scale bars: 10 µm.