Cell composition in BALF of PBS-, LPS-, PLN- and heat inactivated PLN-treated mice.

<p>Macrophage and neutrophil counts in BALF from WT mice, 6 hours after inoculation of PBS, LPS (2 pg/mouse), heat inactivated PLN (HIPLN 500 ng/mouse) or PLN (500 ng/mouse). Data are plotted in Box&Whiskers graph (median+interquartile range N = 5 per group). * P<0.05 versus PBS.</p

PLN activates HEK cells via TLR4.

<p>IL-8 production in HEK-293 cells transfected with CD14 and either TLR2 or TLR4 were incubated with medium (control), LPS (100 ng/ml), LTA (5 µg/ml) or PLN (1 µg/ml) for 6 hours. In some experiments polymyxin B (P) was used at 10 µg/ml. Data are mean±SEM (N = 4 per group). * P<0.01 versus control, † P<0.001 versus control, ‡P<0.001 versus LPS.</p

Inflammatory and cytolytic effects of PLN on mouse alveolar macrophage MH-S cells.

<p>MH-S cells were incubated with increasing doses of PLN for 6 hours with/without polymyxin B (10 µg/ml ) and TNF-α, MIP-2 and cell death were determined thereafter. Cell death was measured using MTT assay as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0007993#s2" target="_blank">Methods</a> section. Data are mean±SEM (N = 5 per group). * P<0.05, † P<0.01 versus control.</p

Roles of TLR2 and TLR4 in lung inflammatory response to high dose PLN <i>in vivo</i>.

<p>Macrophage and neutrophil counts, total protein, IL-6, IL-1β, TNF-α and KC concentrations in BALF from WT, TLR2 KO and TLR4 KO mice, 6 hours after inoculation of 500 ng/mouse. Data are plotted in Box&Whiskers graph (median+interquartile range N = 8 per group). * P<0.05, † P<0.01, ‡ P<0.001 versus WT mice. Dotted line indicates mean value of PBS-treated mice.</p

c-Myc activation is not associated with degree of RHPS4 sensitivity.

<p>(A) c-Myc transcription factor assay. Jurkat cell nuclear extracts show activation and specificity of c-Myc proportional to concentration of extract analyzed and in the presence of wild-type or mutant competitor. (B–C) No significant difference in c-Myc activation was observed between untreated PFSK-1 or C6 cells and RHPS4-treated cells. Asterisk denotes significant reduction in c-Myc levels when either PFSK-1 or C6 untreated cells were exposed to a wild-type oligonucleotide competitor (p≤0.05). (D–E) c-Myc quantitative reverse transcriptase PCR. No difference in PFSK-1 or C6 c-Myc gene expression was observed between representative RHPS4-treated cells and untreated cells.</p

Acute RHPS4 exposure is associated with telomerase inhibition in brain tumor cells <i>in vitro</i>.

<p>(A) TRAP assay using ethanol-precipitated telomere extended DNA products after 30 minutes extension in non-drug treated brain tumor cells. High levels of telomerase activity are observed in each cell line. (B–D) TRAP assays in RHPS4-treated brain tumor lysates reveals complete telomerase inhibition in all cell lines at each drug concentration. 0.1 µg of total protein lysate was loaded per well in each TRAP assay. <i>CHAPS, CHAPS buffer only no lysate control; TS, telomere substrate internal control 61-bp oligonucleotide.</i></p

Acute RHPS4 exposure alters cell cycle dynamics of brain tumor cells <i>in vitro</i>.

<p>PFSK-1 cells exhibited a dose-dependent increase in the proportion of cells in G1-phase. In contrast DAOY, C6 and GB-1 cells exhibited a dose-dependent increase in the proportion of cells in S-phase. PFSK-1 further shows a moderate accompanying increase of sub-G0/1 cells at the higher RHPS4 concentration (5 µM). Percentages are the mean from three independent experiments. Asterisk denotes a significant difference relative to untreated cells.</p

Acute RHPS4 exposure inhibits proliferation of high grade brain tumor cells <i>in vitro</i>.

<p>Proliferation of tumor cells was impaired in malignant brain tumor cells after acute 72 hours exposure to RHPS4. (A–C) PFSK-1, DAOY, U87 and (E) Res196 cells exhibited IC₅₀ values of 2.7, 2.2, 1.1, and 1.6 µM respectively when 0.5–5.0 µM RHPS4 was used, representing a significant inhibition of cell proliferation (p≤0.05 for each drug concentration versus untreated). (D, F–G) Within this concentration range, KNS42, C6 and GB-1 cells were resistant to RHPS4. (H–I) At higher concentrations of RHPS4 exposure C6 and GB-1 cells exhibited IC₅₀ values of 26 µM and 32 µM respectively, representing a significant inhibition of cell proliferation (p≤0.05 for each drug concentration versus untreated). Error bars indicate standard error from three independent experiments. (J–M) Light microscopy of PFSK-1, DAOY, C6 and GB-1 cells showing a marked reduction in cellular density after RHPS4 exposure. <i>Magnifications, x20; Scale bar = 25 </i><i>µm</i>.</p

RHPS4 sensitivity in normal neural and endothelial cells <i>in vitro</i> and <i>ex vivo</i>.

<p>(A) C17.2 cerebellar progenitor cells and (B) HBMEC endothelial cells are sensitive to RHPS4 with an IC₅₀ of 15 µM and 5 µM respectively. (C) Primary rat ependymal <i>ex vivo</i> cultures exhibited functional impairment of ependymal CBF after 3 µM or 30 µM RHPS4 exposure (p≤0.01). (D) A significant reduction in cilia tip distance was observed after either 3 µM or 30 µM RHPS4 exposure (p≤0.01). Error bars represent standard error of the mean from four separate rat brains/experiments.</p

Telomere length measurement and RHPS4-mediated inhibition of Taq polymerase <i>in vitro</i>.

<p>(A) Mean TRF lengths for PFSK-1/DAOY cells (3.8 kb/7.8 kb) and C6/GB-1 (7.5 kb/3.9 kb) cells were determined prior to RHPS4 exposure. (B) Quantitative TRAP assay showing telomerase-mediated telomere extension after 30 minutes (standard TRAP assay) or 5 minutes extension time in non-drug exposed cells. (C) PCR gel showing telomere extension products after 5 minutes extension time in non-drug exposed cells. <i>1, no lysate control; 2–5, PFSK-1, DAOY, C6 and GB-1</i>. (D) PFSK-1 and DAOY showed low levels of telomerase activity at low RHPS4 concentrations and complete absence of activity at high RHPS4 concentrations, when RHPS4 was added pre-telomere extension. C6 and GB-1 showed complete absence of telomerase activity at both RHPS4 concentrations analyzed. (E) Telomerase activity was absent in all cell lines and at both RHPS4 concentrations when RHPS4 was added post-telomere extension. <i>CHAPS, CHAPS buffer only no lysate control; IC, internal control 61-bp telomere substrate oligonucleotide</i>.</p