BC8-15 derivative elevation of progesterone release by MA-10 Leydig tumor cells corresponds with PDE8 inhibitory activity.

<p>MA-10 cells were treated with 20 µM of rolipram, BC8-15, BC8-15A or BC8-15C. Progesterone released into the media after 2 h was measured using a progesterone ELISA kit (Neogen). The data are representative of three independent experiments with 2–4 sample wells for each condition. Values are the mean of four experimental wells ± SEM. Data were analyzed with one-way ANOVA followed by a Tukey’s multiple comparison test (*** p<0.001). The response to BC8-15 is significantly different from that for BC8-15A (p<0.001) and both are significantly different from all other groups (p<0.001).</p

Neutrophils migrate in response to CXCL1 secretion following TLR4 activation in hepatic stellate cells.

<p><b>(A)</b> Schematic representation of the neutrophil chemotaxis assay. <b>(B)</b> Quantification of neutrophil migration in response to secretory WT or TLR4 deficient stellate cells. WT stellate cells were treated (WT HSC + anti CXCL1) or not with anti-CXCL1 antibody. As for internal positive control, the migration of neutrophils towards TLR4 deficient stellate cells supplemented with CXCL1 protein (TLR4 HSC + CXCL1) and with CXCL1 protein only (CXCL1) was quantified in only one experiment. Graphs show three experiments with six mice in each group and statistically significant differences (*<i>P</i><.05) between WT HSCs and TLR4 deficient HSCs, as well as between WT HSCs treated or not with anti-CXCL1, are indicated.</p

BC8-15 elevates testosterone release by primary Leydig cells.

<p>Leydig cells isolated from wild-type (Panel A) and PDE8A^−/−/B^−/− knock-out (Panel B) mice were treated with rolipram (20 µM) or BC8-15 (10 µM and 40 µM) for three hours. Testosterone released into the media was assayed in duplicate for each condition. Values represent the mean ± SEM. Data from wild type and knock-out samples were separately analyzed with one-way ANOVA followed by Tukey’s multiple comparison test. Significant differences are indicated in comparison to the DMSO control (* p<0.05, ** p<0.01, *** p<0.001).</p

Development of a PDE8A inhibitor HTS.

<p><b>A)</b> Structure of PDE4/8 inhibitor BC69 used to optimize PDE8 inhibitor assay. <b>B)</b> A yeast-based screen finds no potent PDE8 inhibitors in a Known Bioactives Collection. PDE8A-expressing strain CHP1204 was screened against 2,640 known bioactive compounds in duplicate wells (plate A and B). Scatter plot of absorbance values from plate A against plate B is shown. Negative control wells contained 0.2% DMSO (black circles), positive control wells contained 10 or 20 µM BC69 (dark gray circles). Experimental wells are shown in light gray. The data set has a correlation value of 0.996.</p

Inhibition of PDE4A, PDE7A and PDE8A by BC8-15 as determined by <i>in vitro</i> enzyme assays.

<p>BC8-15 inhibitory activity was measured by <i>in vitro</i> PDE assays as described in Materials and Methods. Substrate concentrations are 10 nM cAMP for PDE8A and PDE7A and 50 nM cAMP for PDE4A. Each assay was performed at 10 different compound concentrations in duplicate reaction tubes. IC₅₀± SD values are determined by performing non-linear regression analysis on three independent experiments.</p

Summary of the HTS data.

<p>The ability of 220,071 small molecules to promote 5FOA^R growth of a PDE8A-expressing strain was assessed by their composite Z scores (see Materials and Methods). The number of compounds within each indicated composite Z score interval is presented.</p

Hepatic stellate cells are the major source of CXCL1, as shown by both quantification of secretion and <i>in situ</i> localization.

<p><b>(A)</b> Quantification of CXCL1 secretion in enriched fractions of hepatocytes, KCs, LSECs and HSCs, freshly isolated and stimulated <i>in vitro</i> with LPS (1 ng/mL LPS, black squares) during 24 hours. Data are representative of three separate experiments with six mice in each group; ^<b>#</b><i>P</i><.05. <b>(B)</b> <i>In-situ</i> localization of CXCL1 in the liver. Immunofluorescent detection for CXCL1 (red) and liver cells nuclei (blue) for nuclei first shows CXCL1 expression in the sinusoids throughout liver parenchyma. <b>(C)</b> Higher resolution shows that CXCL1 (red) is expressed by sub-endothelial cells, which also store retinol droplets in separate compartments, as shown by CRBP1 staining (green). The Cellular Retinol Binding Protein-1 (CRBP-1) is the best marker to detect simultaneously both resting (Glial Fibrillary Acidic Protein, GFAP+) and activated (α-Smooth Muscle Actin, αSMA+) stellate cells <i>in situ</i>. Alexa Fluor-546-CXCL1 (red) staining does not colocalize either with Tie2-GFP in LSECs (green, <i>upper panel</i>), or F4/80 in KCs (blue, <i>middle panel</i>), but with AlexaFluor-488-CRBP1 (green, <i>lower panel</i>), staining both resting and activated HSCs. TOPRO3 was used for nuclei vizualisation.</p

IC₅₀ values for BC8-15 and related compounds.

<p>The structures of BC8-15 and two structurally-related derivatives are shown. The IC₅₀ values of each compound were determined by <i>in vitro</i> PDE assays (see Material and Methods). The values represent mean IC₅₀± SD determined from three independent experiments.</p

Strain list.

<p>Strain list.</p

Cytokine secretion by hepatocytes, KCs, LSECs and HSCs after isolation from the same liver and in response to low levels of LPS.

<p>Liver cells were freshly isolated on density gradient followed by cell sorting and stimulated with LPS (1ng/mL LPS, black bars or 100ng/mL LPS, hatched bars). Cytokine secretion was measured in the same supernatant with a multiplex assay, run in triplicates. Graphs show three experiments with six mice in each group and statistically significant differences (*<i>P</i><.05) between basal LPS stimulation (1ng/mL) and higher LPS stimulation (100ng/mL) are indicated. Lower panel: bright field images of cells right after isolation (Hepatocytes, LSECs, KCs). Images of HSCs at higher resolution show the retinol droplets at Day 0 and the typical shape of the activated stellate cells after 4 days in culture.</p