1. Volume 8, Issue 6, Pages 492-501 (December 2008)
Macrophage EP4 Deficiency Increases Apoptosis and Suppresses Early Atherosclerosis 
Vladimir R. Babaev, Joshua D. Chew, Lei Ding, Sarah Davis, Matthew D. Breyer, Richard M. Breyer, John A. Oates, Sergio Fazio, MacRae F. Linton 
Cell Metabolism 
Volume 8, Issue 6, Pages (December 2008)
DOI: /j.cmet
Copyright © 2008 Elsevier Inc. Terms and Conditions

2. Figure 1
EP2 Deficiency Causes a Compensatory Increase in EP4 Gene-Expression, and EP4 Deficiency Suppresses COX-2 Production in Peritoneal Macrophages
(A–D) EP4 (A), EP2 (B), COX-1 (C), and COX-2 (D) gene-expression levels in peritoneal macrophages isolated from LDLR−/− mice reconstituted with WT (black), EP4−/− (white), or EP2−/− (gray) FLC and fed with the Western diet for 8 weeks. Macrophages were treated with media alone (control) or with LPS (50 ng/ml) for 5 hr. The gene-expression levels were measured by real-time PCR. Graphs represent data (mean ± SEM) with the same number (n = 3) of mice per group (∗p < 0.05 between control and treated with LPS cells of the same group, and between WT and EP4−/− cells by one-way ANOVA analysis).
(E–F) COX-1 (E) and COX-2 (F) protein levels in peritoneal macrophages. Macrophages were treated with media alone or with LPS for 5 hr. Cell extract (20 μg/line) was resolved on 10% Bis-Tris gel and analyzed by western blot.
Cell Metabolism 2008 8, DOI: ( /j.cmet )
Copyright © 2008 Elsevier Inc. Terms and Conditions

3. Figure 2
EP4 Deficiency in Hematopoietic Cells Does Not Affect Plasma Lipid Levels but Significantly Suppresses Early Atherosclerosis
(A) FPLC profiles in LDLR−/− mice reconstituted WT (black), EP4−/− (white), or EP2−/− (gray) FLCs. The data are represented as the average of total cholesterol of mice (n = 3 per group) reconstituted with different FLC and fed with the Western diet for 8 weeks. Fractions 14–17 contain VLDL, fractions 18–24 are IDL/LDL, and fractions 25–30 contain HDL.
(B–D) Atherosclerotic lesions in aorta en face (B) and extent of atherosclerotic lesion area in the distal (C) and proximal (D) aortas of LDLR−/− mice reconstituted WT (black), EP4−/− (white), or EP2−/− (gray) FLCs. Graphs represent data with different numbers (n = 12, 13, and 13, respectively) of mice of each genotype (p < 0.05 between control and reconstituted with EP4−/− FLC groups by one-way ANOVA analysis, the Tukey test).
Cell Metabolism 2008 8, DOI: ( /j.cmet )
Copyright © 2008 Elsevier Inc. Terms and Conditions

4. Figure 3
EP4 Deficiency in Hematopoietic Cells Increases Apoptosis in Atherosclerotic Lesions
(A) Distribution of TUNEL+ cells in the proximal aorta of mice reconstituted with different FLC and fed with the Western diet for 8 weeks (20x magnification). The scale bars represent 20 μm.
(B and C) Percent of TUNEL+ cells (B) and numbers of DAPI-stained nucleus cells/MOMA-2+ area (C) in atherosclerotic lesions of LDLR−/− mice reconstituted with WT (black), EP4−/− (white), or EP2−/− (gray) FLC and fed with the Western diet for 8 weeks. Graphs represent data (mean ± SEM) with different numbers (n = 12, 13, and 13, respectively) of mice per group (∗p < 0.05 between mice reconstituted with WT and EP4−/− cells by one-way ANOVA analysis).
Cell Metabolism 2008 8, DOI: ( /j.cmet )
Copyright © 2008 Elsevier Inc. Terms and Conditions

5. Figure 4
EP4 Deficiency in Macrophages Increases Susceptibility to Apoptosis and Suppresses Akt and Bad Phosphorylation
(A–D) Detection of TUNEL+ cells in untreated WT (A) and treated with PA-BSA (0.5 mM for 18 hr), WT (B), EP4−/− (C), or EP2−/− (D) macrophages (x20).
(E and F) Percent of TUNEL+ cells in WT (black), EP4−/− (white), or EP2−/− (gray) macrophages untreated or treated with PA-BSA (E), oxLDL (100 μg/ml) or AcLDL (100 μg/ml) plus an ACAT inhibitor, Sandoz (10 μg/ml) (F) for 24 hr. Graphs represent data (mean ± SEM) with the same number (n = 3) of mice per group (∗p < 0.05 between WT and EP4−/− cells by one-way ANOVA analysis).
(G and H) Expression of Akt, p-Akt (serine 473), and p-GSK3α/β (H), p-Bad (serine 136 and 155), and β-actin (J) in WT, EP4−/− or EP2−/− peritoneal macrophages that were untreated or treated with PA-BSA (0.5 mM) for 3, 6, or 18 hr. Cell extract (100 μg/line) was resolved and analyzed by western blot using antibodies against the proteins as indicated.
Cell Metabolism 2008 8, DOI: ( /j.cmet )
Copyright © 2008 Elsevier Inc. Terms and Conditions

6. Figure 5
Inhibition of the PI3K/Akt Signaling Pathway in WT Macrophages Accelerates Apoptosis
(A) Expression of Akt, p-Akt (serine 473), Bad (serine 136 and 155), and β-actin proteins in WT macrophages treated with PA-BSA (0.5 mM) alone or with dibutyryl cAMP (1 mM), RO (100 μM), or PKA inhibitor, H89 (10 μM), for 3 and 6 hr.
(B) Expression of Akt, p-Akt, GKS3α/β and β-actin proteins in WT macrophages treated with PA-BSA alone or with wortmannin (Wrt, 100 nM) for 3 or 6 hr. Extracted proteins (100 μg/lane) were resolved and analyzed by western blot using antibodies against the proteins as indicated.
(C–F) Apoptosis in WT macrophages untreated (C) or treated with Wrt (50 nM; D) or PA-BSA (E) alone or in combination with Wrt (F) macrophages for 18 hr (x20). The scale bars represent 20 μm.
(G) Percent of apoptotic macrophages untreated and treated with Wrt (50 nM; D), PA-BSA alone or in combination with Wrt for 18 hr. Graphs represent data (mean ± SEM) with the same number (n = 3) of mice per group (∗p < 0.05 between untreated cells and treated with PA-BSA alone and with Wrt by one-way ANOVA analysis).
(H–K) Apoptosis in WT macrophages untreated (H) or treated with the Akt inhibitor IV (10 μM; I) or PA-BSA (J) alone or in combination with the Akt inhibitor IV (K) for 24 hr (x20). The scale bars represent 20 μm.
(L) Percent of apoptotic macrophages untreated and treated with the Akt inhibitor IV (10 μM), PA-BSA alone or in combination with the Akt inhibitor IV for 24 hr (∗p < 0.05 between cells treated with PA-BSA alone and with the Akt inhibitor by one-way ANOVA analysis, the Tukey test).
(M) Expression of Akt, p-Akt, and β-actin in WT macrophages treated with Akt inhibitor IV or PA-BSA alone or in combination for 6 hr. Extracted proteins (100 μg/lane) were resolved and analyzed by western blot using antibodies against the proteins as indicated.
Cell Metabolism 2008 8, DOI: ( /j.cmet )
Copyright © 2008 Elsevier Inc. Terms and Conditions

7. Figure 6
EP4 Deficiency in Macrophages Suppresses NF-κB-Related Gene and Protein Expression Levels
(A–I) Expression levels of inflammatory (IL1β, IL6, MCP1, MMP9), NF-κB-related (p-50, IKKα, IKKβ), and antiapoptotic (Gadd45β and Itch) genes in WT (black), EP4−/− (white), or EP2−/− (gray) macrophages. The cells were treated with LPS (50 nM) for 5 hr and gene expression levels were measured by real-time PCR. Graphs represent data (mean ± SEM) of two experiments with the same number (n = 3) mice per group (∗p < 0.05 between WT and EP4−/− cells by one-way ANOVA analysis).
(J) Protein expression levels in WT and EP4−/− macrophages treated with PA-BSA (500 μM) for 3 and 6 hr. Cell extract (100 μg/line) was resolved and analyzed by western blot. The graph represents the data of two different experiments.
Cell Metabolism 2008 8, DOI: ( /j.cmet )
Copyright © 2008 Elsevier Inc. Terms and Conditions

8. Figure 7
A Schematic Presentation of the EP2 and EP4 Receptor-Signaling Pathways Specific for Mouse Peritoneal Macrophages Relevant to Apoptosis
Cell Metabolism 2008 8, DOI: ( /j.cmet )
Copyright © 2008 Elsevier Inc. Terms and Conditions