CAS, HuR and nucleolin provide appropriate references for the 3D reconstruction of nucleoli.

<p>(A) HeLa cells were stained with antibodies against, HuR, nucleolin and CAS as described for Fig. 1. A z-stack was acquired and a single slice of the stack is depicted. Size bar is 20 µm. (B) The z-stack was processed with Imaris (Bitplane) software to generate isosurfaces. Images are shown for HuR (red), nucleolin (green) and CAS (magenta). Bottom panels show the combination of either HuR and nucleolin (left) or CAS and nucleolin (right).</p

DEM and DRB change the protein composition of nucleoli.

<p>HeLa cells were treated with vehicle, DEM or DRB as detailed in Materials and Methods. Nucleolar pixel intensities for B23 and nucleolin were quantified for the same nucleoli. CAS and nucleolin were combined as markers for samples treated with ethanol or DEM. CAS alone provided a reference for cells incubated with DMSO or DRB. Multiple independent experiments were analyzed for DEM (4 data sets) and DRB (3 data sets). Segmentation images were inspected visually to eliminate misidentified nucleoli. Nucleolar fluorescence was then measured for at least 30 cells for each condition and experiment. Results are normalized to controls and depicted as average +SEM; *, p < 0.05 or **, p < 0.01. Size bar: 20 µm.</p

HuR is not a suitable nucleolar marker in heat stressed cells.

<p>HeLa cells were heat-shocked and immunostained for HuR and nucleolin. Nucleolin outperformed HuR for the compartment identification during heat shock and at 1 hour of recovery. After longer periods of recovery (2 and 3 hours), however, HuR identified nucleoli properly. Size bar: 20 µm.</p

CAS and HuR, but not nucleolin, delimit nucleoli in DRB-treated HeLa cells.

<p>Cells were incubated with (A) DMSO or (B) DRB essentially as described <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0080237#pone.0080237-Kodiha1" target="_blank">[21]</a> and processed as in Fig. 2. Individually, the marker proteins CAS and HuR detected nucleoli upon DRB incubation, although some nucleoli were missed (indicated by arrow heads). The identification of nucleoli was improved by combining the information from CAS and HuR images with the <i>add</i> function <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0080237#pone.0080237-Kodiha1" target="_blank">[21]</a>. Nucleolin was redistributed by DRB throughout the nucleoplasm. Based on the nucleolin image, neither the “detect light holes” nor “detect dark holes” filter could identify nucleoli. Size bar is 20 µm.</p

Nucleolin, CAS and HuR provide markers to demarcate nucleoli in oxidant-treated HeLa cells.

<p>HeLa cells were incubated with (A) the vehicle ethanol or (B) the oxidant DEM as described <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0080237#pone.0080237-Mahboubi1" target="_blank">[36]</a>. Immunostaining of CAS, HuR and nucleolin was carried out as for Fig. 1, and nucleoli were identified based on these markers. Segments generated for CAS, HuR or nucleolin and their overlays are shown on the right side. Note that nucleolin was superior to demarcate nucleoli in DEM-treated cells. While CAS and HuR were useful to delineate nucleoli, this occurred with reduced accuracy. Non-identified nucleoli are marked with arrowheads; size bar is 20 µm.</p

Nucleolin, CAS and HuR identify the nucleolus in HeLa cells treated with actinomycin D.

<p>HeLa cells were incubated with (A) the solvent DMSO or (B) 100 nM actinomycin D according to ref. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0080237#pone.0080237-Kodiha1" target="_blank">[21]</a>. Following treatment, samples were processed as in Fig. 2, and confocal images were used to identify nucleoli. Comparison of the segments and their overlay reveals that in control and actinomycin D-treated cells, all of the three proteins served as appropriate markers for nucleoli. Size bar is 20 µm.</p

Nucleolin, CAS and HuR are suitable to detect nucleoli in actinomycin D-treated MCF7 cells.

<p>MCF7 cells were treated with (A) DMSO or (B) 100 nM actinomycin D. Cells and images were processed as described for Fig. 2. Size bar: 20 µm.</p

Markers that identify nucleoli under various experimental conditions.

<p>Nucleolin, CAS and HuR were assessed with the software operations described in Materials and Methods. Specifically, we evaluated the ability of each candidate to generate segments that colocalize with black or bright holes, respectively. This assessment was performed for the different experimental conditions listed.</p>1<p>For DRB treatment, the most accurate demarcation of nucleoli was obtained when CAS and HuR were combined as markers.</p

DEM, actinomycin D and DRB inhibit <i>de novo</i> RNA synthesis in nucleoli.

<p>HeLa cells were incubated with vehicle, DEM, actinomycin D or DRB as described in Materials and Methods. 5<i>-</i><b>Ethynyluridin</b>e (EU) was present during the last hour of the incubation period, and cells were processed for the detection of EU, CAS and nucleolin <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0080237#pone.0080237-Kodiha5" target="_blank">[42]</a>. Three independent experiments were performed for each compound. Misidentified nucleoli were removed from the analysis by visual inspection. Nucleoli of at least 30 cells were quantified for each condition and experiment. Data were normalized to controls and shown as average +SEM. Significant differences are indicated by *, p < 0.05 or **, p < 0.01. Size bar: 20 µm.</p

CAS, HuR and nucleolin demarcate nucleoli in 3D.

<p>Images for isosurfaces depicted in Fig. 10 were sliced in two different planes. The panels display the results for HuR (red), nucleolin (green) and CAS (magenta). Overlays show the combination of either HuR and nucleolin or CAS and nucleolin.</p