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

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

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

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

Effect of phenformin, resveratrol and AICAR on the abundance of nucleolar proteins and histone H3 phosphorylation.

<p>(A) Kidney cells were treated with vehicle or the pharmacological compound indicated. Crude extracts were probed with antibodies against B23, fibrillarin, nucleolin or RPA194; actin provided a loading control. The abundance was calculated as nucleolar protein/actin for at least three independent experiments; results were defined as 1 for control samples. Data are shown as averages+SEM; significant differences are marked with *<i>p</i><0.05 or **<i>p</i><0.01. (B) Signals for phospho(Ser10)-histone H3 (p-H3) were measured for crude cell extracts as described for part A.</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

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

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

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

Pharmacological AMP Kinase Activators Target the Nucleolar Organization and Control Cell Proliferation

<div><p>Aims</p><p>Phenformin, resveratrol and AICAR stimulate the energy sensor 5′-AMP activated kinase (AMPK) and inhibit the first step of ribosome biogenesis, <i>de novo</i> RNA synthesis in nucleoli. Nucleolar activities are relevant to human health, because ribosome production is crucial to the development of diabetic complications. Although the function of nucleoli relies on their organization, the impact of AMPK activators on nucleolar structures is not known. Here, we addressed this question by examining four nucleolar proteins that are essential for ribosome biogenesis.</p><p>Methods</p><p>Kidney cells were selected as model system, because diabetic nephropathy is one of the complications associated with diabetes mellitus. To determine the impact of pharmacological agents on nucleoli, we focused on the subcellular and subnuclear distribution of B23/nucleophosmin, fibrillarin, nucleolin and RPA194. This was achieved by quantitative confocal microscopy at the single-cell level in combination with cell fractionation and quantitative Western blotting.</p><p>Results</p><p>AMPK activators induced the re-organization of nucleoli, which was accompanied by changes in cell proliferation. Among the compounds tested, phenformin and resveratrol had the most pronounced impact on nucleolar organization. For B23, fibrillarin, nucleolin and RPA194, both agents (i) altered the nucleocytoplasmic distribution and nucleolar association and (ii) reduced significantly the retention in the nucleus. (iii) Phenformin and resveratrol also increased significantly the total concentration of B23 and nucleolin.</p><p>Conclusions</p><p>AMPK activators have unique effects on the subcellular localization, nuclear retention and abundance of nucleolar proteins. We propose that the combination of these events inhibits <i>de novo</i> ribosomal RNA synthesis and modulates cell proliferation. Our studies identified nucleolin as a target that is especially sensitive to pharmacological AMPK activators. Because of its response to pharmacological agents, nucleolin represents a potential biomarker for the development of drugs that diminish diabetic renal hypertrophy.</p></div