Comparison of imidazole derived inhibitor SB203580 with the dibenzosuberone derived inhibitor Skepinone-L in the MK2-EGFP translocation assay.

<p>(A) Nucleocytoplasmic intensity ratios of activated U2OS MK2-EGFP cells after incubation with increasing concentrations (0 µM –100 µM) of SB203580 (blue) or Skepinone-L (red) Incubation with Skepinone-L induces an increased nucleocytoplasmic ratio of <1 up to 10 nM of compound concentration indicating a strong inhibitory effect on p38 MAPK. (B) Representative cellular images of the selected data points (scale bar: 20 µm).</p

Structures of identified top 10 inhibitors.

<p>Structures of identified top 10 inhibitors.</p

Phospho-Ser63-c-Jun/JNK kinase assay in living cells.

<p>(Left) representative pictures of phospho-Ser63-c-Jun antibody signal in the nucleus upon pathway activation. (Right) Top ten lead inhibitors identified from the MK2-EGFP translocation screen have negligible effects on JNK/phosphor-c-jun activity at relevant concentrations (<15 µM). Significant effects (* p<0.05) for several p38 inhibitors are detected at very high concentrations (> = 150 µM). As positive control a specific JNK inhibitor SP600125 is shown. SD derived from 3 independent experiments.</p

Validation of the screening results using the MK2-EGFP translocation assay at decreasing compound concentrations.

<p>Confirmation of p38 inhibition of selected 378 cherry-picks at (A) 1.5 µM, (B) 150 nM, and (C) 15 nM. Green stripe: Skepinone-L top level bench mark of inhibition of nuclear export of MK2-EGFP. Yellow strip: nuclear intensity in activated cells treated with DMSO. Top 40 inhibitors are shown, including Skepinone-L and negative control (DMSO). (D) Comparison of lead inhibitor activity at 1.5 µM, 150 nM and 15 nM. Nucleocytoplasmic fluorescent ratio after treatment with Skepinone-L is set to 1. For direct comparison nucleocytoplasmic fluorescent ratios induced by all validated compounds are normalized to Skepinone-L.</p

Activation of p38 MAPK in response to certain stimuli.

<p>Dashed lines refer to one or more stimuli. Bold arrows refer to translocation of the kinase in response to activation by upstream kinases.</p

Determination of cellular toxicity of top ten lead inhibitors.

<p>Analysis of metabolic viabilities using the AlamarBlue assay after incubation of the cells with the top ten p38 inhibitors for 24 µM. Incubation with DMSO serves as negative control. Shown is standard deviation derived from 3 independent experiments.</p

Cellular MK2-EGFP translocation assay to screen for p38 inhibitors.

<p>(A) Schematical outline of MK2-EGFP translocation assay. Nuclear DAPI segmentation facilitates determination of the nucleocytoplasmic MK-2-EGFP fluorescence intensity ratio (grey ring). In the ground state U2OS cells stably expressing MK2-EGFP shows a slightly higher fluorescent intensity in the nucleus compared to the cytoplasm. For activation of the Rac/p38 pathway cells were subjected to hyperosmotic stress (175 mM NaCl/350 mOsm) leading to a rapid relocation of MK2-EGFP from the nucleus to the cytoplasm. Co-treatment with p38 inhibitors abolishes this redistribution, which is visualized in high content analysis. Determination of fluorescent intensities provides a cellular read-out with appropriate z’ values above 0.5. (B) Representative pictures of activated U2OS MK2-EGFP cells either non-treated (DMSO) or treated with p38 inhibitors Skepinone-L or SB203580, scale bar 20 µM. (C) Workflow of the p38 inhibitor screen, validation procedures and number of identified compounds (D) Sorted results of the entire primary p38 inhibitor screen represented as average cellular nucleocytoplasmic fluorescence intensity ratios reflecting the distribution of MK2-EGFP (E) Rank comparison of the first and second biological replica of the primary screen at 15 µM showing the reproducibility of the results. R² of >0.85 indicate reliability for further cherry-pick listing.</p

Comparative data table of top 35 inhibitors selected from MK2-EGFP translocation assay (TA) determining the nuclearcytoplasmic ratio at 15 nM compound concentration (N = 3), whole blood assay (WBA) determining TNFα release after LPS stimulation (N = 3) and the p38 MAPK activity assay (BAA) determining the IC50 on p38 MAPK activity detected by inhibition of phosphorylation of ATF2 (Thr69/17) (N = 3).

<p>Comparative data table of top 35 inhibitors selected from MK2-EGFP translocation assay (TA) determining the nuclearcytoplasmic ratio at 15 nM compound concentration (N = 3), whole blood assay (WBA) determining TNFα release after LPS stimulation (N = 3) and the p38 MAPK activity assay (BAA) determining the IC50 on p38 MAPK activity detected by inhibition of phosphorylation of ATF2 (Thr69/17) (N = 3).</p

FRAP analysis of subcellular chromobody populations.

<p>(<b>A</b>) Fluorescence recovery of CA_NTDcb1eGFP over time, either expressed alone (upper panel) or in combination with HIV-1 Gag (lower panel). Arrows in the lower panel indicate two regions of interest: Diffusely distributed CA_NTDcb1eGFP signal in the cytoplasm and focal structures at the outer cellular rim, indicating virion assembly sites at the membrane. Scale bar, 10 µm. (<b>B</b>) Quantitative FRAP analysis reveals different mobility states of unbound and ubiquitous, diffusely and cytoplasmic as well as focal and membranous CA_NTDcb1eGFP signal. Errors are standard 0deviations (n≥7). (<b>C</b>) 3D-FRAP representation of the same cell shown in (A) (lower panel), illustrating cytoplasmic recovery to almost initial level, while cumulated, membranous signal shows no recovery.</p

Visualizing the formation of HIV-1.

<p>A HeLa-Kyoto cell, expressing CA_NTDcb1eGFP and HIV-1 Gag was monitored for 90 min at 1 min time intervals using confocal spinning disk microscopy (see also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0050026#pone.0050026.s001" target="_blank">Movie S1</a>). Shown are projections of 12 z-sections per time point. Scale bar, 10 µm.</p