The Pab 1801-positive puncta contain PB components.

<p>The indicated p53+/+ or −/− cell lines were simultaneously immunostained with Pab 1801 and antibodies specific for the PB components Dcp1a, Rck/p54, Hedls or Xrn1. In all cases, all the cytoplasmic <i>foci</i> recognized by the Pab 1801 were also recognized by the antibodies against PB markers. Bars, whole cells, 10 µm; magnifications, 1 µm.</p

RNase treatment or knockdown of specific PB components provokes the simultaneous dissolution of PBs and Pab 1801 puncta.

<p>A, live U2OS cells were permeabilized with 0.1% TX100 for five minutes and then exposed to CSKB without (Control) or with 100 µg/ml of RNase (RNase) for additional 5 minutes. The percentage of cells with PBs simultaneous stained with Dcp1a and Pab 1801 was evaluated in 100 cells from duplicate experiments for each treatment and diminished from 75% in control cells to 40% in RNAse-treated cells. The remaining Pab 1801 foci have less than half of the size of control cells. B, U2OS and SK-N-SH cells were treated with specific siRNAs against the PB components Hedls, Rck/p54 or 4ET, and stained with the indicated antibodies. The Pab 1801 puncta vanished when PB are disrupted. The proportion of cells with <i>foci</i> is indicated, as evaluated in 100 cells from duplicate experiments for each treatment. As before (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036447#pone-0036447-g004" target="_blank">Figures 4</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036447#pone-0036447-g005" target="_blank">5</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036447#pone-0036447-g006" target="_blank">6</a>), all puncta were double stained for each pair of antibodies and single-stained <i>foci</i> were not present in any of the treatments. Bar, 10 µm.</p

Pab 1801 puncta are present in p53-null cells.

<p>A, B, p53+/+ or p53−/− HCT116 cells were treated with daunorubicin and immunostained with the Pab 1801 (A) or the Pab DO1 (B). With both antibodies, a nuclear signal is observed upon stimulation of p53+/+ cells but not p53−/− cells. Pab 1801-positive puncta are always present in p53+/+ and p53−/− cells independently of p53 levels. C, p53-null H1299 cells were treated with the indicated drugs and stained with the Pab 1801. The Pab 1801 puncta disappeared upon exposure to cycloheximide and remained unaltered after puromycin treatment. The percentage of cells with punctate Pab 1801 signal was evaluated in 100 cells from duplicate stainings for each treatment. Bar, 10 µm. D, alignment of human and rat p53 N-terminus including the Pab 1801 epitope (grey box). The 10-aa epitope is absent in the rat sequence. E, Hippocampal rat neurons were prepared as described <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036447#pone.0036447-Baez2" target="_blank">[27]</a> and stained with the Pab 1801 or the Pab DO1. A merge of the immunofluorescence and the phase contrast images is shown. Pab 1801-positive puncta are detected in cell soma and dendrites. Bars, whole cells, 10 µm; magnifications, 1 µm.</p

Pab 1801 puncta and PBs are simultaneously induced by different strategies.

<p>A, B, U2OS cells were treated with two different combinations of arsenite and puromycin: 250 µM arsenite +100 µg/ml puromycin (1× induction) and 500 µM arsenite +250 µg/ml puromycin (2× induction). A, The average number of PBs per cell increased from 4 in control cells to 14 in treated cells, as evaluated in 100 cells from duplicate experiments for each treatment. Induced PBs were stained with the Pab 1801 in all cases. An example of the 2× induction is shown in Ars+Puro panel. B, the size of Dcp1a-foci and Pab 1801 foci increased at the same rate and in a dose-dependent manner. Median values of more than 100 foci size for each treatment were plotted. C, overexpression of the translational repressor Smaug 1 also provoked 1801 foci induction. U2OS cells were transfected with Smaug 1-ECFP and number of foci per cell and foci size were analized in transfected (T) and neighbouring non-transfected cells (NT). The average number of PBs per cell increased from 5 in non-transfected cells to 55 in transfected cells, as evaluated in 100 hundred cells of each condition. In all cases PBs were stained with the 1801 antibody. The foci size increased more than twice, as evaluated for both 1801 and Rck/p54 in 200 non-transfected foci and 750 Smaug1-ECFP transfected foci. Bars, 10 µm.</p

Commercial and domestic sources of Pab 1801 behave similarly.

<p>A, U2OS p53+/+ and H1299 p53−/− were simultaneously stained with Dpc1a, Rck/p54 and Pab 1801 from a commercial source (left panels) or from domestic preparations of hybridome supernantant (right panels). B, HCT 116 p53+/+ and HCT116 p53−/− were simultaneously stained with Hedls and Pab 1801 from the same two sources. The strong pattern of Pab 1801 puncta was similar for both commercial and domestic sources of the Pab 1801 in p53+/+ and p53−/− cell lines. Bars, whole cells, 10 µm; magnifications, 1 µm.</p

The Pab 1801 does not recognize <i>Drosophila</i> PBs, and stains a subset of PBs in rat neurons.

<p>A, S2R+ cells were immunostained with the Pab 1801 and an antibody against Ge-1/Hedls. No signal of the Pab 1801 was observed. Bars, 10 µm. B, rat primary neurons were simultaneously stained with Pab 1801, Hedls and Tubulin β III. Pab 1801 puncta and Hedls colocalize partially. Bars, whole neurons, 10 µm; dendrites, 1 µm. C, representative dendritic fragments showing immunostaining against Pab 1801 and the PB components Xrn1, Dcp1a and Rck/p54. As above, Pab DO1 yielded no signal. Bars, 1 µm.</p

Different procedures give the same immunostaining pattern of Pab 1801.

<p>U2OS cells were treated as indicated and immunostained with Pab 1801 and Hedls. A, U2OS cells were fixed as usual in 4% PFA. B, live U2OS cells were treated with methanol-acetone during 3 minutes at room temperature (mild non-aq). C, live U2OS cells were treated with methanol-acetone during 20 minutes at −20°C (strong non-aq). D, live U2OS cells were permeabilized with 0.1% Triton X-100 for 10 minutes prior to fixation in PFA 4%. Nuclear staining with Pab 1801 looks stronger than in control cells. None of these treatments affected the cross-reaction of the Pab 1801 with PBs. Bars, whole cells, 10 µm; magnifications, 1 µm.</p

The Pab 1801 yields a cytoplasmic punctate pattern.

<p>U2OS, WI38, SK-N-SH, and HCT116 cells were fixed in PFA, permeabilized in Triton 0.1% and immunostained with the indicated antibodies against p53 as indicated in Materials and Methods. A, Under resting conditions, the Pab 1801 showed a cytoplasmic granular pattern, whereas the FL 393, the Pab 240, the Pab 421 and the Pab DO1 antibodies yielded a faint nuclear signal. B, WI38 cells were treated with daunorubicin or actinomycin D, and immunostained with the indicated antibodies. A strong p53 nuclear signal was observed in all cases. The Pab 1801 punctate signal in the cytoplasm remained unaltered. Bar, 10 µm.</p

PBs and Pab 1801-positive puncta simultaneously dissolve upon polysome stabilization.

<p>U2OS cells were treated with CHX or PURO during one hour, and immunostained with the indicated antibodies. The Pab 1801 punctate signal vanished together with the PBs upon cycloheximide treatment. Both <i>foci</i> remained intact upon exposure to puromycin. The percentage of cells with PBs simultaneous stained with Dcp1a and Pab 1801 was evaluated in 100 cells from duplicate experiments for each treatment. The Pab 1801 <i>foci</i> were never observed free of Dcp1a, and PBs were always stained with the Pab 1801. Bar, 10 µm.</p

Summary of manual and BUHO analysis for the identification of SG regulators.

<p>S2R+ cells were treated with the indicated dsRNA and exposed to arsenite. SG formation was analyzed in 7 images (test set) with an average of 300 cells for each treatment. Manual values and BUHO generated results are indicated.</p