Effects of SP on LPS-mediated macrophages phagocytosis and migration.

<p>(A) RAW 264.7 cells were incubated with SP (10 μM) and/or LPS (100 ng/ml) for 24 hours and neutral red phagocytosis was evaluated by adding dye for 1 hour at 492 nm. Data are means ± SD. ** p<0.01 versus untreated. The morphology of the treated cells were visualised by Zeiss microscopy at a 20 x magnification. (B) RAW 264.7 cells were plated on the upper chamber; SP (10 μM) and LPS (100 ng/ml) were used as a chemoattractant in the lower chamber. DHMEQ (2.5 μg/ml) was used as p65 nuclear translocation inhibitor. After 24 hours, quantification of transwells migration was evaluated by counting five random fields per treatment well and averaged across two independent experiments by light microscopy (20 X). The results are presented as migration percent in relation to the total number of untreated cells. Values are means ± SD. ** p<0.01 versus untreated or versus each drugs alone.</p

SP induces HO-1 mRNA and protein expression.

<p>RAW 264.7 cells were treated with the indicated concentrations of SP for (A) 4, (B) 6 and (C) 24 hours and mRNA expression levels were assessed by RT-qPCR. (B). The results shown are the means ± SD of two experiments (each of which was performed in triplicate). *p<0.05 and **p<0.01 <i>versus</i> each agent alone. (D) Cells were treated with SP (μM) for 24 hours. The induction of HO-1 protein expression (32 kDa) was analysed by western blotting. The numbers represent the fold of HO-1 difference with untreated control samples (Ctrl) arbitrarily set at 1.0. The data represent two independent representative experiments.</p

HO-1 induction promotes M2 macrophage polarisation.

<p>RAW 264.7 cells were transfected with siRNA HO-1 and siRNA NC (non-correlated). (A) RT-qPCR analysis of HO-1 mRNA in cells transfected 48 hours after transfection. Cells were treated with SP (10 μM) and/or LPS (100 ng/ml) for 24 hours and mRNA expression of selected genes was evaluated by RT-qPCR (B) IL-6 mRNA expression (C) TNF-α mRNA expression (D) Arg1 mRNA expression (E) IL-10 mRNA expression and (F) Rel A mRNA expression. The data are mean ±SD of two separate experiments, each of which was performed in triplicate. **p < 0.01 versus siRNA NC.</p

Effects of SP on LPS-induced p65 transcription factor nuclear translocation.

<p>(A) RAW 264.7 cells were treated with or without SP (10 μM) and/or with LPS (100 ng/ml) for 4 hours and cytoplasmic and nuclear fractions were separated and analysed by western blotting for NF-κB p65 expression. We used β-actin (47 kDa) and lamin B1 (67 kDa) immunolabelling as loading controls for cytoplasmic and nuclear fractions, respectively. The numbers represent the fold difference of NF-κB p65 with untreated control (Ctrl) arbitrarily set at 1.0. The data shown represent two independent experiments with comparable outcomes. (B) RT-qPCR analysis of p65 (Rel A) mRNA in cells treated or untreated with SP (10 μM) for 4 hours. The data are mean ±SD of two separate experiments, each of which was performed in triplicate. **p < 0.01 versus untreated. (C) Immunofluorescence analysis of RAW 264.7 cells with NF-κB p65 (red) in cells treated with or without SP (10 μM) and LPS (100 ng/ml) for 4 hours. The nuclei of the cells have been counterstained with DAPI (blue). Scale bar 20 μm. Results are shown from two independent experiments with comparable outcomes.</p

SP induces M2-like phenotype.

<p>(A) RAW 264.7 cells were treated with SP (10 μM) and Arg1, IL-10 and IL-4 mRNA expression was evaluated with RT-qPCR. Data are the means ± SD of two experiments (each of which was performed in triplicate). *p<0.05 and **p<0.01 <i>versus</i> control. (B) RAW 264.7 cells were treated with SP (10 μM) for 24 hours and Arg1 (35 kDa) protein levels were determined by western blotting. Representative immunoblots of results obtained from three independent experiments are shown. (C) RAW 264.7 cells were treated with different doses of SP or stimulated with LPS 100 ng/ml for 24 hours. NO in the culture medium was measured as described in the methodology section. Data are expressed as nitric oxide concentration (μM) and are the means ± SD of three separated experiments, each of which was performed in triplicate. *p<0.05 versus untreated.</p

Comparison of common and distinct gene expressions across the various differentially- expressed gene groups in HepG2 and Huh7 cells upon celecoxib and sorafenib treatment.

<p>(A) Venn diagram analyses of genes, differentially expressed (≥2-fold) in HepG2 and Huh7 cell lines upon CLX (50 µM) treatment, SOR (7.5 µM) treatment, and combined SOR+CLX treatment. (B) Venn diagram comparison of common and distinct genes uniquely modulated (≥2-fold) in HepG2 and Huh7 cells only following combined SOR+CLX treatment. (C) Hierarchical clustering based on the 174 genes list (2-fold difference in gene expression; <i>p</i>-value cutoff of 0.05) which discriminates HepG2 and Huh7 cells according to their response to combined SOR+CLX treatment. Red signifies up-regulation and green signifies down-regulation.</p

Selected differentially expressed (≥2-fold) functional gene groups in HepG2 and Huh7 cells upon combined sorafenib+celecoxib treatment.

<p>Selected differentially expressed (≥2-fold) functional gene groups in HepG2 and Huh7 cells upon combined sorafenib+celecoxib treatment.</p

IPA functional pathway analyses of genes differentially expressed (≥2-fold) in HepG2 and Huh7 cell lines upon combined SOR+CLX treatment.

<p>IPA functional pathway analyses of genes differentially expressed (≥2-fold) in HepG2 and Huh7 cell lines upon combined SOR+CLX treatment.</p

Network analysis of dynamic gene expression in Huh7 cells based on the 2-fold common gene expression lists obtained following combined SOR+CLX treatment.

<p>The four top-scoring networks have been merged and are displayed graphically as nodes (genes/gene products) and edges (the biological relationships between the nodes). Figure legends are as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0065569#pone-0065569-g006" target="_blank">Figure 6</a>.</p

Effect of celecoxib (CLX) and sorafenib (SOR) individually and in combination on apoptosis and cell proliferation.

<p>(A) Detection of apoptosis by TUNEL assay. Photomicrographs of HepG2 cells treated for 24 h with the indicated concentrations of CLX and SOR either alone or in combination. Apoptotic cells were visualized by TUNEL staining as described in the Materials and Methods section. (B) Quantitative analysis of TUNEL-positive HepG2 and Huh7 cells. Data are expressed as the means ± SD of two separate experiments. *<i>p</i><0.05, versus each agent alone. (C) Cell proliferation was assessed by BrdU assay. Cells were treated for 48 h with the indicated concentrations of CLX and SOR either alone or in combination. Data are expressed as the percentage of the control cells and are the means ± SD of three separate experiments. *<i>p</i><0.05; **<i>p</i><0.01 versus each agent alone.</p