Supplementary Figure 3: Actin dynamics but not transcriptional inhibition affect early G1 nuclear expansion. Detection of Flag-actin derivatives by doxycycline-inducible T2A-SNAP fusions.
From: A transient pool of nuclear F-actin at mitotic exit controls chromatin organization
(a) 3D surface reconstructions of NIH3T3 nuclei (vizualized by SiR-DNA) at indicated times after drug treatment at mitotic exit. Scale bar, 10 μm; time stamp, hours:min. (b) Nuclear volume quantifications in cells treated similar to a. Data is shown as mean ± s.d. (n = 50 nuclei per condition, pooled from at least 3 independent experiments). (c) Design of Flag-tagged nuclear actin derivatives linked to the SNAP-tag by a T2A peptide. Upon translation, the T2A peptide is cleaved resulting in equimolar expression of Flag-NLS-actin and the SNAP-tag. Accordingly, labelling of the SNAP-tag allows for indirect detection of Flag-NLS-actin in living cells. (d) Immunoblot confirms doxycycline-induced expression of Flag-NLS-actin-T2A-SNAP derivatives. A single band indicates efficient cleavage of Flag-NLS-actin-T2A-SNAP. (e) Confocal images of fixed NIH3T3 cells expressing nAC-GFP transfected with Flag-NLS-actin-T2A-SNAP derivatives. In contrast to Flag-NLS-actin the SNAP-tag (labelled by SiR-647) displays pancellular distribution. Scale bar, 10 μm. (f) Confocal images of fixed NIH3T3 cells at mitotic exit show expression of Flag-NLS-actin-T2A-SNAP derivatives, as indicated. Magnifications correspond to dashed rectangles and highlight Flag-actin. Scale bar, 10 μm. (g) Nuclear volume quantifications in live NIH3T3 cells stably expressing H2B-mCherry and doxycycline-induced BFP-NLS, NLS-BFP-actinR62D or Flag-NLS-actin-T2A-SNAP derivatives. Expression of the indicated constructs was induced during G0. Data are mean + s.d. from n = 30 nuclei per condition. (h) RT-qPCR analysis of FOS expression in serum-starved NIH3T3 cells, pre-treated with Flavopiridol (1 μM for indicated times) before stimulation with serum (20% FCS, fetal calf serum) for 30 min. Note that 15 min pre-treatment with Flavopiridol efficiently inhibits serum-induced transcriptional upregulation of FOS mRNA. Data are shown as mean from n = 2 independent experiments. (i) Nuclear volume analysis in NIH3T3 cells stably expressing H2B-mCherry in the presence of Flavopiridol (1 μM) or DMSO (0.1%). Flavopiridol was added at metaphase prior to imaging the subsequent expansion of daughter nuclei. Data are shown as mean ± s.d. (n > 14 nuclei [precise n?] per condition, pooled from 3 independent experiments). Unprocessed original scans of blots are shown in Supplementary Fig. 7.
