The distribution of genes for different pathways in the gene coexpression network modules. The 3,600 most varying genes were used for a weighted gene coexpression network construction and subjected to GO biological process pathway analysis using the EASE software [37]. Values shown are the percentage of pathway genes present in the coexpression network that are clustered in color-labeled network modules. The colors correspond to the color-labeled modules defined in Figure 3a.

DEP and ox-PAPC induce HO-1 expression in HMEC. (a) Western blot. HMEC were treated with DEP, ox-PAPC or a combination of both at various concentrations. Mouse monoclonal anti-HO-1 and anti-β-actin antibodies were used to detect the relevant proteins as described in Materials and methods. (b) Densitometric analysis. The expression level of HO-1 protein in optical density (OD) units is shown. Similar levels of β-actin are shown in (a). Results are typical of one representative experiment (n = 4).

DEP and ox-PAPC induce a large number of genes in HMEC. (a) Experimental protocol. HMEC were treated in triplicate wells with DEP, ox-PAPC, or DEP + ox-PAPC at the various concentrations shown. Cells were harvested at 4 h and cytoplasmic RNA prepared. Illumina microarrays were run and the data confirmed by qPCR analysis of selected genes. (b) Venn diagrams of upregulated genes. The numbers of genes that were significantly upregulated (> 1.5-fold, p < 0.05) over controls (no treatment) by the various treatment are shown. The left Venn diagram summarizes the number of genes induced by DEP 5 μg/ml (DEP5), ox-PAPC 10 μg/ml (ox10) and DEP5 + ox10. The middle Venn diagram shows the number of genes induced by DEP5, ox-PAPC 20 μg/ml (ox20) and DEP5 + ox20. The right Venn diagram summarizes the number of genes induced by DEP5, ox-PAPC 40 μg/ml (ox40) and DEP5 + ox40. The total number of genes induced by a particular condition can be found by adding all values displayed within the circle corresponding to that condition. Values displayed in the circle intersections indicate the number of genes induced in common by the intersecting conditions.

Ambient ultrafine PM chemicals enhance in vivo expression of genes related to vascular inflammation. (a) Experimental protocol. Two-month-old male apoE null mice fed a high-fat diet were exposed for a total of 120 h (5 h/day, 3 days/week for 8 weeks) to concentrated ultrafine particles (UFP), concentrated PM_2.5 (FP), filtered air (FA) or not exposed (NE). (b) Hepatic gene expression levels. Gene expression was determined by qPCR of mRNA prepared from liver homogenates. UFP-exposed mice exhibited marked upregulation of HO-1 (left), XBP1 (center) and ATF4 (right). Values were normalized by β-actin mRNA levels and expressed as fold control (FOC). Five samples per group were assayed in duplicates. Statistical analysis was performed by one-way ANOVA, Fisher PLSD.

DEP and ox-PAPC co-regulate genes in a synergistic/additive fashion. (a) Synergistic index (SI). Synergy was defined as the presence of a co-regulatory effect by both DEP and ox-PAPC that was greater than the effects induced by either compound alone and greater than the sum of those individual effects. The following criteria for a synergistic effect were as follows: SI (ΔAB/(ΔA+ΔB) > 1; AB (mean) > A (mean), p ≤ 0.05; AB (mean) > B (mean), p ≤ 0.05, where ΔA is the difference in mean expression level between the DEP and the control samples, ΔB is the difference in mean expression level between the ox-PAPC and the control samples, and ΔAB is the difference in mean expression level between the DEP + ox-PAPC and the control samples. (b) mRNA expression levels of representative genes. Each graph displays the relative mRNA expression levels normalized by β₂-microglobulin mRNA levels and expressed as fold control (FOC) for microarray (white bars, left-hand y-axis) and qPCR (black bars, right-hand y-axis) assessment of representative genes (HO-1, IL-8, ATF4, CXCL1, XBP1, IL-11). For ease of comparison, the qPCR scale was divided by factors of 3.5 (HO-1) and 3 (IL-8), respectively. In similar fashion, the microarray scale was divided by a factor of 4 (IL-11) to make the comparison easier. The asterisk indicates combinations of DEP + ox-PAPC that exhibited synergistic effects. The high consistence of microarray and qPCR analysis, conducted on triplicate samples from independent experiments, implies both technical and biological validation. Statistical analysis was performed by one-way ANOVA, Fisher PLSD.

Gene coexpression network analysis. (a) The gene coexpression network. The 3,600 most varying genes were selected to construct a weighted gene coexpression network. I, The average linkage hierarchical clustering tree; II, clustered gene modules represented by different colors; III, gene significance of the individual modules. The green, brown and yellow modules were enriched in significant genes most highly correlated with the treatment conditions (p < 0.0001). Gene significance = -log (p value). IV, The synergistic gene enrichment. The mean synergistic indices (SI) of network genes that were upregulated by DEP, ox-PAPC and the corresponding combinatorial treatment of DEP plus ox-PAPC were calculated for each network module. The green, brown and yellow modules were also enriched in genes synergistically coregulated. Mean SI, mean synergistic index as defined in Materials and methods. (b) Heat map of the green module; (c) Heat map of the brown module. Expression levels of (b) 307 genes and (c) 426 genes are represented in the rows by color coding (green = low expression, red = high expression), in triplicate samples for each treatment condition (columns). Both modules show a clear synergistic/additive pattern where the combinatory treatments exhibited as a whole either a greater level of upregulation (towards red) in 274 genes (b) and 335 genes (c) at the top or downregulation (towards green) in 33 genes (b) and 91 genes (c) at the bottom, compared with the corresponding concentrations of DEP and ox-PAPC alone. Color scale is shown at the right of both heat maps, ranging from 0 (indicated by the green color at the bottom) to 1.0 (indicated by the red color at the top) as a reflection of the level of mRNA expression. DEP5 and DEP25, DEP 5 and 25 μg/ml, respectively; ox10, ox20, ox40: ox-PAPC 10, 20 and 40 μg/ml, respectively; DEP5 + (ox10, ox20 ox40): DEP 5 μg/ml + ox-PAPC 10, 20 and 40 μg/ml respectively.