SUPPLEMENTAL MATERIAL

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1 SUPPLEMENTAL MATERIAL Cell Selective Cardiovascular Biology of Microsomal Prostaglandin E Synthase- Chen, et al Cell specific role of mpges- Lihong Chen,,*, MD, PhD; Guangrui Yang,,*, MD, PhD; Xiufeng Xu 3, PhD; Gregory Grant,, PhD; John A. Lawson,, BS; Mohammad Bohlooly-Y 3, PhD and Garret A. FitzGerald,, MD, FRS, Institute for Translational Medicine and Therapeutics,, Department of Pharmacology, University of Pennsylvania, Philadelphia, PA, 94 3, RAD-Transgenics, Discovery Sciences, Astra Zeneca R&D, Mölndal, 4383 Mölndal, Sweden * These authors contributed equally to this work. Corresponding Author: Garret A. FitzGerald Institute for Translational Medicine and Therapeutics, Department of Pharmacology, University of Pennsylvania, - Translational Research Center, 34 Civic Center Blvd, Philadelphia, PA, 94 Fax: , Tel: , garret@upenn.edu. The journal subject code: [3] [6] [7]

2 Supplemental Methods. Targeted disruption of mpges- To construct a mpges- gene targeting vector, 4. kb,.9 kb and.8 kb genomic DNA fragments were prepared from a BAC clone which contains about kb 9/Sv genomic sequence including the mpges- gene (ResGen, Invitrogen Corporation) for 5 homology, deletion and 3 homology sequences respectively. The fragments were cloned into a plasmid containing loxp-loxp-pgkneo-loxp sequence, applying a standard cloning technique. In brief, a.9 kb fragment containing exon of mpges- was inserted between an isolated loxp site and loxp-flanked PGK-Neo cassette and then placed between the two homology sequences. The targeting vector was linearized and electroporated into R mouse embryonic stem (ES) cells and ES cell clones were selected with G48 (3 µg/ml). The candidate ES cell clones underwent homologous recombination were screened by PCR and confirmed by Southern blotting according to a standard protocol. The targeted ES cells were injected into C57BL/6J blastocysts and the injected embryos were implanted into the uteruses of pseudopregnant B6CBA mice. Chimeric males were mated with C57BL/6 females to produce agouti heterozygous animals (F). The heterozygous F males were bred with Rosa6Cre transgenic mice on a C57BL/6 background to delete the whole loxp floxed region, including the exon and the neo cassette. mpges- null mice were then obtained by intercrossing. Offspring were genotyped by PCR using primers 8, 83 and 84 (8, forward: 5'- AAGGTTGATGGTGCACGTCTA-3 ; 83, reverse: 5 -TTTGTTTGGCCTCACTCTACA-3 ; 84, forward: 5 - CTCAGGCAGGGCTCATCTACC-3 ) that detected wild type alleles (.79 kb fragment) and null alleles (.47 kb fragment), respectively. For generating mpges- conditional mice, the heterozygous F mice were bred with EIIaCre transgenic mice on a C57BL/6 background to

3 delete the loxp flanked PGK-Neo cassette only, keeping the loxp flanked.9 kb deletion fragment in the genome. The genotyping for conditional mice was achieved by PCR using primers 8 and 83 to identify the wild type (.79 kb fragment) and floxed alleles (.399 kb fragment), respectively. Three primers located in different exons, 854, 855 and 856 (854, forward: 5'- CCAGGTGCTCCCGGCCTTTCT-3', 855, reverse: 5'-CGCTCCACATCTGGGTCACTC-3'; and 856, reverse: 5'-GTAGGCCACGGTGTGTACCAC-3') were used for RT-PCR analysis. A.7 kb fragment can be amplified by RT-PCR from wild type, but not from KO mice with primers 854 and 855. Applying primers 854 and 856,.33 kb or.39 kb fragments can be obtained by RT-PCR from wild type and KO mice, respectively.. Immunohistochemistry After conventional deparaffining and hydration, sections were treated with 3% H O for min to eliminate endogenous activity of peroxidase. Then sections were heated in a target retrieval solution (DakoCytomation, Carpinteria, CA) for 3min. After cooling at room temperature and washing with PBS, sections were incubated with primary antibodies against α-sma, PCNA (Abcam, Cambridge, MA), TN-C (Chemicon International Inc., Temecula, CA) or CD45 (Abcam, Cambridge, MA) for 6min followed by incubation with antibody enhancer and Polymer-horseradish peroxidase conjugated secondary antibodies and chromogen AEC staining (Golden Bridge International, Mukilteo, WA). After several washes with distilled water, sections were counterstained with hematoxylin and mounted routinely. 3. Endothelial cell characterization The identification of mouse endothelial cells was confirmed by staining for platelet endothelial cell adhesion molecule (PECAM-) and by DiI-Ac-LDL uptake as previously described. Briefly,

4 the cells were passaged onto sterile glass coverslips and grown overnight. For PECAM- staining, the cells were incubated with or without mouse anti-pecam- monoclonal antibody (:) for hour at room temperature after being fixed for minutes in acetone. Then they were washed with PBS and incubated with rhodamine-conjugated secondary antibody (:) for hour. After extensive washing, the cells were visualized with a fluorescence microscope. For the acetylated LDL uptake experiments, the cells were incubated with μg/ml DiI-Ac-LDL (Biomedical Technologies Inc., Stoughton, MA) in DMEM for hour at 37 C. Then the medium was removed and the cells were washed with PBS and the DiI-Ac-LDL was visualized with a fluorescence microscope.

5 A Wild-type allele S.6 kb 8 83 S Targeting vector PGK neo 4. kb.9 kb.8 kb DTA S 7.5 kb S Targeted allele PGK neo Cre In vivo Probe Conditional allele 8 83 Cre In vivo Deleted allele Supplemental Figure

B 5 4 3 9 8 7 6 5 4 3 C 854 855 856 5 UTR 3 3 UTR cdna -/- +/- +/+ -/- +/- +/+ -/- +/- +/+.

6 B C UTR 3 3 UTR cdna -/- +/- +/+ -/- +/- +/+ -/- +/- +/+.6 kb 7.5 kb mpges- (854/855) 7bp mpges- (854/856) 3bp 39bp GAPDH Muscle Lung Brain Supplemental Figure

7 A BM VD B mpges- 6kd β-actin 4kd flox TieCre/flox Supplemental Figure

8 A Fluorescence Phase PECAM- No primary antibody B Uptake of Dil-Ac-LDL Supplemental Figure 3

9 IL-β Relative mrna level 3 * Mac-mPGES--WT Mac-mPGES--KO ctrl LPS4h Supplemental Figure 4

10 A EP EP3 B EP EP3 3 flox SM/flox Tie/flox flox SM/flox Tie/flox 3 WT KO.3... WT KO EP EP4 EP EP4 5 5 flox SM/flox Tie/flox 4 3 flox SM/flox Tie/flox WT KO WT * KO Supplemental Figure 5

11 Supplemental Figure Legends Supplemental Figure. Targeted disruption of the mpges- gene. A, The native 5 region of the mpges- gene and targeting vector (top), the targeted and conditional allele (middle) and the disrupted mpges- gene (bottom) are shown. A region containing exon was flanked by loxp sites and was deleted after breeding with Cre transgenic mice. The open rectangles with numbers indicate the exons. The filled rectangle shows the untranslated region (UTR). Horizontal bars indicate the probe used for Southern blotting. DTA, diphtheria toxin A fragment gene cassette. S, SpeI. The triangle indicates loxp site. The arrow indicates the oligos used for genotyping. B, Southern blot analysis of the targeted ES clones. Genomic DNA was digested with SpeI and probed with a 3 flanking probe shown in (A). Expected sizes of DNA fragments of the wild-type and mutant alleles are indicated in (A). Lane and, markers; Lane 3-3, targeted clones. Lane 4-5, wild type ES clones. C, mpges- transcript (top) and RT-PCR analysis (bottom). Tissues were obtained from mpges- +/+, +/- and -/- littermates. The arrows indicate the oligos used for RT-PCR. Supplemental Figure. Western blot analysis of mpges- expression in bone marrow cells of the TieCre mice. Total protein of bone marrow (BM) cells was extracted from mpges-- flox and TieCre/flox mice. Vas Deferens (VD) was used as a positive control for mpges- expression and β-actin staining was performed as a protein loading control. 4µg protein of each sample was loaded. A) Representative blots. B) Quantitative analysis of mpges- protein expression (normalized to β-actin) (n=3). Supplemental Figure 3. Characterization of mouse ECs. A, The ECs isolated from the mouse aorta were examined for the presence of the endothelium-specific marker PECAM-. Fluorescence and phase images show that the positive PECAM- staining was observed in

12 almost all of the cells. Images with no primary antibody were used as background controls. B, ECs were identified by the DiI-Ac-LDL uptake experiments. Fluorescence and phase images show that almost all cells are able to uptake DiI-Ac-LDL after hour incubation. Supplemental Figure 4. Inflammatory cytokine expression in mpges- deleted macrophages. Real-time RT-PCR of IL-β expression in peritoneal macrophages. Thioglycollate-elicited macrophages harvested from Mac-mPGES--WT (WT) or Mac-mPGES- KO (KO) mice were treated with or without 5μg/ml LPS for 4h and whole cell lysates were prepared for quantitative RT-PCR analysis. All samples were normalized to 8s rrna (n=3; *P=.5, nonparametric Mann-Whitney test). Supplemental Figure 5. Expression of EP receptors in wire-injured femoral arteries. A, Real-time RT-PCR analysis of the relative expressions of EP, EP, EP3 and EP4 in injured arteries from SMCre/flox, TieCre/flox mice and mpges--flox mice 8 days after wire injury(n=4-5). B, Real-time RT-PCR analysis of the relative expressions of EP, EP, EP3 and EP4 in injured arteries from Mac-mPGES--WT or Mac-mPGES--KO mice 8 days after wire injury (n=5; *P<.5, unpaired t-test with welch s correction).

13 Supplemental References. Shi W, Haberland ME, Jien ML, Shih DM, Lusis AJ. Endothelial responses to oxidized lipoproteins determine genetic susceptibility to atherosclerosis in mice. Circulation. ;:75-8.

A Low salt diet. C Low salt diet + mf4-31c1 3. D High salt diet + mf4-31c1 3. B High salt diet

A Low salt diet. C Low salt diet + mf4-31c1 3. D High salt diet + mf4-31c1 3. B High salt diet A Low salt diet GV [AU]:. [mmhg]: 0..... 09 9 7 B High salt diet GV [AU]:. [mmhg]: 7 8...0. 7.8 8.. 0 8 7 C Low salt diet + mf-c GV [AU]:. [mmhg]:.0.9 8.7.7. 7 8 0 D High salt diet + mf-c E Lymph capillary