Cellular growth edge effect reduction.

<p>Cell-lines were processes as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0105598#pone-0105598-g002" target="_blank">Figure 2</a> using crystal violet. Mean p-values +/− SEM comparing borders versus core for different parental cell-lines at 0 or 1 h room temperature incubation are reported.</p

HTS using former protocol.

<p>A. Screening of 768 clones (8 plates) of a metagenomic library for PPARγ modulation using HT-29 reporter gene assay (RGA). Bacterial clone lysates were applied on HT-29-PPARγ (10% vol/vol) for 24 h and luciferase activity (RLU) was monitored. Border, core and control wells are represented in grey, white and red dots respectively. B. HT-29-PPARγ-luc, SW1116-ANGPTL4-luc and HT-29-NFkB-SEAP were seeded in 96 well-plates and homogeneously activated with sodium butyrate (2 mM, for PPARγ and ANGPTL4) or <i>E. coli</i> lysate (for HT-29-NFkB-SEAP) using a pipetting robotic workstation. 24 h after activation, luciferase (for PPARγ and ANGPTL4) and SEAP (for NFκB) activities were monitored. Border and core wells are represented in grey and white dots respectively. p-values indicated on the figure were obtained by a unpaired t-test of core <i>versus</i> border values.</p

Cellular growth-dependent edge effect reduction applied to three different reporter cell-lines.

<p>Cell-lines were processes as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0105598#pone-0105598-g003" target="_blank">Figure 3</a>. The table represents the mean of three p-values +/− SEM for comparing borders versus core for the different tested reporter cell-lines.</p

Detection limit on optimized reporter gene assay.

<p>The HT-29-PPARγ cell-line was homogeneously seeded using a pipetting robotic workstation and incubated at room temperature for 1 h prior to 37°C incubation. After 24 h of culture, all wells were activated homogenously with EPI300-Cont lysates for 24 h at 37°C. In randomly chosen wells (50% border and 50% core), three different concentrations of PPARγ activating NaBut (at 500 µM, 160 µM and the sub-activating concentration 80 µM) were randomly added in the 96 well-plates. The graph represents normalized values from 3 independent plates.</p

Different normalizations of assay plates.

<p>Different mathematical approaches for normalization were applied to the dataset from the initial HT-29-PPARγ screening. A shows all data-points as dots ordered by activity-rank whereby the red dots are points on the plate’s cores and the green points are well on the borders. Dark green areas show overlapping of red and green dots. B shows ranked Z-score normalization of the results. (red dots are points on the cores and the green points are well on the borders. Dark green areas show overlapping of red and green dots. C. shows the ranked B-score values for the same data set. (red dots are points on the cores and the green points are well on the borders. Dark green areas show overlapping of red and green dots.</p

Metagenomic clones growth optimization.

<p>Four random plates of EPI300 metagenomic clones were inoculated from their −80°C glycerol stock into 96 well-plates (10% v/v) using a pipetting robotic workstation. After a 24 h pre-culture, they were re-inoculated and cultured overnight (10% v/v) at 37°C. These steps were performed either in static or agitated (700 rpm) culture conditions. Bacterial growth was monitored by optical density (OD 600 nm). A. Border and core wells of bacterial cultures in agitated (left panel) or static culture-conditions (right panel) for thus representative set of 4 plates. p-values indicated on the figure are the result of a unpaired t-test of the core <i>versus</i> border bacterial culture ODs. B. Growth curves of one representative plate of metagenomic clones (OD 600 nm) for agitated and static culture conditions. Red dots: mean OD600, joined grey dots: growth curve of single metagenomic clones.</p

PPARγ HTS screening.

<p>Comparison of different data analysis methods of screening data from the screening of 92×96 metagenomic clones on a PPARγ RGA. Figure A represents Z-score normalized data B represents B-score normalized data. The top left picture plots the each readout in function of the growth of the metagenomic clone. The bottom left picture represents the distribution of the readouts over the whole dataset and the large picture on the right part represents all data points ranked by readout. The coloration of red dots for the borders and empty green dots for the core allow to visualize eventual position effects.</p

Schematic overviewed of challenges addressed and the resulting suggested protocol.

<p>Schematic overviewed of challenges addressed and the resulting suggested protocol.</p

Detection limit of the screening protocol.

<p>Statistical normalization applied to optimized cellular and bacterial culture conditions. As in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0105598#pone-0105598-g005" target="_blank">Figure 5</a>, HT-29-PPARγ cell-line was homogeneously seeded using a pipetting robotic workstation and incubated at room temperature for 1 h prior to incubation. After 24 h homogenous EPI300-Cont lysates were applied to the cell assay (1/10 v/v) for 24 h at 37°c. In randomly chosen wells (50% border and 50% core), three different concentrations of PPARγ activating NaBut (at 500 µM, 160 µM and the sub-activating concentration 80 µM) were randomly added in the 96 well-plates. The resulting measured luciferase activity (expressed in relative light units, RLU) is represented as activity-ranked dots. Luciferase activation for EPI300-Cont, EPI300-Cont+500 µM NaBut, EPI300-Cont+160 µM NaBut and EPI300-Cont+80 µM NaBut are represented in black, red, green and blue respectively. Grey bars represent mean activation (Mean) and mean activation +/− standard deviation (SD).</p

Edge effect reduction on cellular growth applied to the HT-29-PPARγ reporter cell-line.

<p>A Parental HT-29 cells were homogeneously seeded using a pipetting robotic workstation and pre-incubated at room temperature for the indicated time (0, 0.5 or 1 h) prior to 37°C incubation for 24 h. Cellular monolayer homogeneity was monitored using cell staining with crystal violet and quantified by absorption measurement at 595 nm. Left panel is a surface plot of a representative set of plates at increasing times of room temperature pre-incubation. Boxplot representation (right panels) of the OD 595 values for the border compared to the core of the respective representative plates. Mean p-values for different cell-lines tested are summarized in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0105598#pone-0105598-t001" target="_blank">Table 1</a>. B HT-29-PPARγ reporter cells were homogeneously seeded using a pipetting robotic workstation and pre-incubated at room temperature for the indicated time (0 or 1 hour) then at 37°C for 24 h prior to sodium butyrate (2 mM) activation. Luciferase activity (RLU) was quantified after 24 h of activation. Graph (left panels) shows two representative plates with different pre-incubation times at room temperature. Boxplots (right panels) represent the border and core values of the surface plots. Mean p-values for different cell-lines tested are summarized in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0105598#pone-0105598-t002" target="_blank">Table 2</a>.</p