Activated protein kinases related to UP induced apoptosis.

<p><b>A</b>. Effects of exposure of P388 cells to 0.05 µM UP for 1 h on the phosphorylation status and expression level of protein kinases. The expression was quantified using the computerized image analysis system ImageQuant. Each column represents those scanned in duplicate of three wells expressed as percentage of control ± Standard Deviation. ***, <i>P</i><0.001, UP vs. Control. <b>B</b>. Effects of MAPK inhibitors on UP-induced P388 growth inhibition,the cells were pretreated with U0126 (10 µM), SP60012 (20 µM) or SB203580 (20 µM) for 1 h, then co-treated with UP for 72 h. U0126, ERK inhibitor; SP60012(SP), JNK inhibitor; SB203580(SB), P38 inhibitor. **, P<0.01, PG vs.(PG+inhibitor),UP vs.(UP+ inhibitor).</p

Dicarabrones A and B, a Pair of New Epimers Dimerized from Sesquiterpene Lactones via a [3 + 2] Cycloaddition from Carpesium abrotanoides

Dicarabrones A and B, a pair of epimers possessing a new skeleton featuring a cyclopentane ring connecting two sesquiterpene lactone units, were isolated from the whole plant of Carpesium abrotanoides L. Their full structures were established on the basis of spectroscopic data and were further confirmed by single-crystal X-ray crystallography. They were presumably biosynthesized from two sesquiterpenoid monomers through a [3 + 2] cycloaddition. Dicarabrones A and B showed moderate effects on HL-60 cells with IC₅₀ values of 9.1 and 8.2 μM, respectively

Acidification does not participate in UP caused growth inhibition.

<p>A. Acridine orange staining of P388 cells, PG and MP were 0.05 µM, AO was 5 µg/ml. Orange granules were acidified compartments. B. Represent data of flow-cytometric pHi analysis using 10 µM BCECF-AM staining after the cells were incubated with 0.05 µM of PG or UP for 24 h with or without imidazole (0.5 mM) pretreatment for 1 h. a, Control; b, PG; c, PG+imidazole; d, UP; e, UP+ imidazole C. Effect of acidification inhibitor on UP-induced P388 growth inhibition, the cells were pretreated with imidazole (0.5 mM ) for 1 h, then co-treated with PG (0.05 µM) and UP (0.05 µM) for 72 h.</p

Effect of UP on the growth of P388 cells.

<p>Cells were treated with indicated concentration of UP and PG for 72 h. The data shown represent the percentages of cell viability of three independent experiments with three determinations in each. **, <i>p</i><0.01 and ***, p<0.001 vs. Control.</p

UP causes ROS generation, which is most likely not involved in UP-induced apoptosis.

<p>A. P388 cells were treated with 0.05 µM UP for 1 h, and stained with DCFH (10 µM). Fluorescence was measured by flow cytometry. a, DMSO+DCFH; b, 0.05 µM PG; c, 0.05 µM PG +DCFH; d, 0.05 µM UP; e, 0.05 µM UP+DCFH. B. P388 cells were pretreatment with or without NAC (0.5 mM) for 1 h, then cells were co- incubated with PG (0.05 µM ), UP (0.05 µM) and H₂O₂ (0.4 mM) for 72 h. *, P<0.05, H₂O₂ vs (H₂O₂+NAC).</p

Structures of prodigiosin (PG) and Undecylprodigiosin (UP).

<p>Structures of prodigiosin (PG) and Undecylprodigiosin (UP).</p

UP induces apoptosis and G2/M phase arrest in P388 cells.

<p>A. DNA histograms of P388 cells cultured in medium alone, or in DMSO (1∶1000) or in the presence of 0.05 µM PG and UP for 24 h. B. Expression of apoptosis-related proteins was determined by immunoblotting with specific antibodies following exposure to UP for 24 hr. The expression was quantified using the computerized image analysis system ImageQuant. Each column represents those scanned in duplicate of three wells expressed as percentage of control ± Standard Deviation. *, <i>P</i><0.05, UP vs. Blank. C. Representative flow cytometry profiles by Rhodamine 123 staining, after the cells were incubated with 0.05 µM of UP for 24 h. a, DMSO; b, 0.05 µM PG; c, 0.05 μΜ UP.</p

Ribosome proteins binding to UP identified by MS.

<p>Ribosome proteins binding to UP identified by MS.</p

UP binds to the ribosome.

<p><b>A</b>. The strip of UP in Native–PAGE. <b>B</b>. Sucrose density gradient fractionation of UP-treated P388 cells and UP alone. <b>C and D.</b> Subcellular localization of Rps3 and UP in P388 cells (C) and A549 cells (D) detected by confocal. UP fluorescence is shown in red, Rps3 fluorescence in green and the colocalization (merge) in yellow.</p

The inhibitory action of MdOS on angiogenesis.

<p>A, effect of MdOS on growth factor-stimulated proliferation of human microvascular endothelial cells (HMECs). HMECs proliferation was assayed as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0003774#s4" target="_blank">Materials and Methods</a>. B, effect of MdOS against HMEC tube formation on Matrigel. HMECs were plated on Matrigel in medium with various concentrations of MdOS for 8 h. <i>Left</i>, representative photographs (100×) of three independent experiments. <i>Right</i>, quantification of the inhibitory activity of MdOS on tube formation. C, effect of MdOS on microvessel outgrowth arising from rat aortic ring. Aortic rings were embedded in Matrigel in 96-well plates, then fed with medium containing various concentrations of MdOS for 6 days. <i>Left</i>, representative photographs (100×) of three independent experiments. <i>Right</i>, the area of microvessels was quantified and normalized to untreated controls. D, effect of MdOS on angiogenesis in a chorioallantoic membrane model. Glasscover-slip saturated with MdOS or normal saline was placed areas between preexisting vessels in the fertilized chicken eggs and incubated for 48 h. <i>Left</i>, representative photographs (100×) of three independent experiments. The Glasscover-slip was placed on the right side of the field. <i>Right</i>, the number of vessel branches was quantified and normalized to untreated controls. Data shown are mean±SD from three independent experiments.*, P<0.05; **, P<0.01, versus control.</p