Minimal inhibitory concentrations (MIC in µg/ml) of the isolates AA, BB, C, D and control.

<p>Isolates were tested against skin pathogens like <i>MRSA</i>, <i>Propionibacterium acnes</i> and <i>Staphylococcus epidermidis</i> in a classical MIC dilution assay to define the lowest concentration for <i>in vitro</i> inhibition. Therefore, the compounds or fractions were diluted between 250 and 0.48 µg/ml and overlaid with bacterial solution (10⁴ bacteria/ml). The table indicates that the isolated compound C shows the best activity against all tested bacteria.</p

Minimal inhibitory concentrations (MIC in µg/mL) of the <i>T. wortmannii</i> compound C in comparison with antibiotics used for acne treatment on different <i>P. acnes</i> strains.

<p>Compound C, minocycline, erythromycin and doxycycline were tested in a 96 well dilution assay under anaerobic conditions and the minimal inhibitory concentration (MIC) defined 24–48 h later as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0097929#pone-0097929-t001" target="_blank">table 1</a>. Tests were done in triplicates and the table shows the average value of multiple MIC tests.</p

Analytical HPLC/MS profiles.

<p>(A) HPLC/MS profile of the crude extract of <i>T. wortmannii</i> isolated with EtOAc. (B) Chromatogram after straight phase and sephadex-G50 separation.</p

Effect of <i>T. wortmannii</i> components on <i>P. acnes</i>-induced IL-8 expression.

<p>(A) HaCaT cells were pre-treated with 20 µg/ml components AA, BB, C, D, Crude extract or left untreated for 2 h and IL-8 production stimulated with <i>P. acnes</i> suspension for 24 h. Control experiments were run with cells alone (n.c.) or cells stimulated with <i>P. acnes</i> suspension (pos. contr.). (B) Pre-, co-, and post-treatment effects of several concentrations of component C on <i>P. acnes</i>–induced IL-8 expression in HaCaT cells. (C) HaCaT cells were stimulated with different <i>P. acnes</i> strains (<i>P. acnes</i>, <i>P. acnes</i> (ERY^R), <i>P. acnes</i>_5-7) and post-treated with 20 µg/ml component C 3 h later. Data are presented as the mean+- standard deviation of three independent experiments. Statistical significance is indicated by *, p<0.05; **, p<0.01, Students <i>t</i> test.</p

Effect of <i>T. wortmannii</i> substance C on <i>P. acnes</i>-induced activation of NF-

<p>κ<b>B and AP1.</b> HaCaT cells were transiently transfected with NF-κB- (A) or AP-1- Luciferase (B) as described in Fig. 3C and 24 h later treated with 0, 10, 20 or 40 µg/ml component C. After 2 h cells were then stimulated by incubation with <i>P. acnes</i> suspension and luciferase activities measured 8 h later. The data are the mean of three experiments +− standard deviation. (C) HaCaT cells were pre-treated with component C (Sub. C) for 2 h, and then stimulated with <i>P. acnes</i> suspension for 20 min. Phosphorylation and total protein expression of ERK, JNK, and IkB-α were detected by western blot analysis using specific antibodies. Tubulin was used as a loading control.</p

Screening for anti-inflammatory <i>T. wortmannii</i> components.

<p>(A) Flow cytometric analysis of ICAM-1 expression on the cell surface of HUVECs. Cells were stimulated with 10 ng/ml hTNF-α with or without 25 µg/ml crude extract or left untreated as negative control and stained with mouse anti-human ICAM-1 mAB. (B) Screening for anti-inflammatory substances, using a cell-based ELISA. Cells were treated for 1h in the absence or presence of 25 µg/ml substances AA, BB, C, D and crude extract, prior to being stimulated for 24 h with 10 ng/ml hTNF-α and stained with human ICAM-1 mAB (C) HEK293 cells were transiently transfected with NF-κB-Luciferase construct and EGFP construct. 24 h later, transfected cells were treated with 0, 40, 20 or 10 µg/ml substances or crude extract, prior to being stimulated with 10 ng/ml hTNF-α. Luciferase activities was determined 8 h later, normalized to the EFGP activities and expressed as fold increase over the control. (*, p<0.05; **, p<0.01, Students <i>t</i> test).</p

Cytotoxicity of <i>T. wormannii</i> extract and isolates.

<p>(A) HUVECs were plated at 10⁶ cells/ml on goldfilm electrods in 96 well plates and allowed to attach and form a monolayer for 24 h before cells were treated with 1, 10 or 100 µg/ml crude extract or 20% DMSO (control) or left untreated and cell toxicity measured every 5 min for 24 h. (B) HeLa, CaCo-2, HaCaT, HUVEC and HKER cells were plated at 2×10⁵ cells/ml in 96 well plates for 6 h and stimulated with 500 µg/ml–0.002 µg/ml crude extract for 24 h before Alamar blue was added (10%) and fluorescence intensity measured and the IC₅₀ determined. (C) HUVECs were treated with 20 µg/ml or 200 µg/ml crude extract, with staurosporine (positive control) or left untreated (neg. control) for 24 h and apoptotic and necrotic cells measured using AnnexinV and 7-AAD. HaCaT (D), HUVEC (E) and HKER (F) were plated as described in B and cells stimulated with AA, BB, C and D.</p