Supplementary Figure 4: Inhibition of nuclear F-actin formation impairs chromatin decompaction at mitotic exit.
From: A transient pool of nuclear F-actin at mitotic exit controls chromatin organization
(a) Quantification of GFP-H2B fluorescence lifetime in cells expressing GFP-H2B alone, or in combination with mCherry-H2B and indicated treatments: trichostatin A (TSA), or sodium azide (NaN3) together with 2-deoxyglucose (2-DG). ∗∗P < 0.01, ∗∗∗P < 0.001 calculated by one-way ANOVA. (b) Quantification of fluorescence lifetime reveals no significant difference in GFP fluorescence lifetime upon expression of and staining for Flag-Exportin 6 using a Alexa Fluor 405-conjugated antibodies. ns, non-significant in one-way ANOVA. (c) Comparative immunoblot analysis of histone modifications (H3S10ph, H4K16ac) in NIH3T3 cells induced to express BFP-NLS or NLS-BFP-actinR62D and undergoing either asynchronous (asyn.) or synchronized (mitotic shake off) culture, as indicated. (d,e) Images and quantitative immunofluorescence analysis of nuclear Aurora B (d) and KAT5 (e) (both green; nuclei are stained with DAPI (magenta)) in NIH3T3 cells at mitotic exit expressing Flag-NLS-actin-T2A-SNAP derivatives, as indicated. Data are shown as mean ± s.d. (n = 20 nuclei per condition, pooled from three independent experiments). Scale bar, 10 μm. ∗∗∗∗P < 0.0001 calculated by t-test. (f) Analysis of chromatin compaction by an MNase accessibility assay 45 min after mitotic shake-off in NIH3T3 cells expressing either doxycycline-induced Flag-NLS-actinwt or -actinR62D. Graph shows quantified pixel intensities corresponding to band intensities. (g) Example images illustrating the pipeline used for the quantification of condensed chromatin in cryo-electron microscopy images (for details see Methods). Based on raw images (I) nuclei and nucleoli were manually segmented (II). Condensed chromatin was semi-automatically segmented across the nucleoplasmic region and classified (III) allowing for an assessment of its distribution using a custom ImageJ/Fiji macro (IV). (h) Representative electron microscopy images of cryo-preserved, synchronized early G1 NIH3T3 cells expressing GFP-Exportin 6 corresponding to Fig. 4l. Scale bar, 2 μm. Unprocessed original scans of blots are shown in Supplementary Fig. 7. Immunoblot in c and MNase accessibility assay in f represent 1 out of 2 independent experiments.
