The Role of P38a MAPK in Atherosclerosis Figure 5. Similar plaque characteristics, cytokine, chemokine and adhesion molecule expression in p38aMY-KO/ApoE2/2 and ApoE2/2 mice. Staining and quantification at the aortic sinus of p38aMY-KO/ApoE2/2 and ApoE2/2 mice for: (A) Collagen content, Masson Trichrome staining (blue fibres, indicated by arrows), (B) Foam cell content, MOMA2 staining (red, indicated by arrows), (C) Necrotic core area, clear areas in lesions that did not stain for toluidine blue (nec = necrosis). (D) Relative mRNA levels of adhesion molecules, pro-inflammatory cytokines and chemokines (left to right) of aortal arches from p38aMY-KO/ApoE2/2 and ApoE2/2 mice after 10 weeks on HCD. P38aMY-KO/ApoE2/2 females, n = 8; ApoE2/2 females, n = 9. Scale bar 50 mm. Error bars represent SD. doi:10.1371/journal.pone.0021055.g005 OxLDL stimulation of PMs was performed as previously described, using 50 mg/ml DiI-labeled oxLDL [31]. Foam cell formation was visualized by staining overnight with Oil-red O (red) and counterstaining with hematoxylin. Uptake of oxLDL was quantified by flow cytometry (PE). For MLECs, before oxLDL stimulation, cells were starved overnight unless otherwise indicated and 100 mg/ml oxLDL was added to the medium for indicated timepoints. Immunoblot analysis Immunoblot analysis was performed as described elsewhere [29]. Membranes were probed with specific antibodies against a- tubulin (Sigma-Aldrich, T6074), p38a (Cell signaling, #9218), and p-p38 (Cell Signaling, #9211). Horseradish peroxidase-conjugated anti-rabbit and anti-mouse secondary antibodies were used (Amersham). Band volumetric analysis on western blots was performed using Radames software (Kindly provided by Dr. Hanns-Eugen Sto¨ ffler). P38a and total phosphorylated p38 were calculated by dividing the abundance of each protein by tubulin abundance (Figure S4). Southern blot analysis Genomic DNA isolated from BMDM was digested overnight at 37uC in a 50 ml BamHI reaction (16BamHI restriction buffer, 1 mM spermidine, 1 mM DTT, 100 mg/ml BSA, 50 mg/ml RNase A and 50 U BamHI enzyme/reaction). The DNA fragments were separated on a 0.8% agarose gel that was denatured by soaking for 45 mins in denaturing solution (0.4 M NaOH, 0.7% NaCl), and transferred overnight by capillary flow onto the surface of a charged nylon membrane (Hybond XL, Amerscham). The membrane was then soaked in neutralization solution (0.5 M Tris-HCl, 1.5 M NaCl), incubated at 70uC for 1 hr and prehybridized in hybridization buffer (1 M NaCl, 50 mM Tris-HCl pH 7.5, 10% dextransulphate, 1% SDS, 250 mg/ml Salmon Sperm DNA) at 65u for 3 hrs. DNA probes labeled by random priming with 32a-dGTP were added to the hybridization buffer and allowed to hybridize to the membrane overnight at 65uC. The membrane was then washed and exposed to autoradiographic film. Probe sequences used for the detection of p38a wild-type, floxed and deleted DNA fragments are available upon request. Quantitative real-time PCR RNA was isolated from aortas, macrophages and endothelial cells using Trizol-reagent (Invitrogen) and RNeasy columns (QIAGEN), as previously described [7]. Briefly, RNA (1 mg) was used for reverse transcription with SuperScript III reverse transcriptase (Invitrogen). The reaction was topped up to 200 ml with water, and 2 ml were used for quantitative real-time PCR (qRT-PCR) reaction with the TaqMan qPCR Kit from Eurogentec. Standardization was performed with GAPDH (TaqMan). Taqman probes used for the quantification of MCP-1,VCAM1, IP-10, Gro-KC, RANTES, MIP-1a, Fractalkine, MIP-1b, MCP-3, IL-1b, IL-18, TNF, IL-6 and IL-10, were obtained from Applied Biosystems. Statistical analysis All statistical analyses were performed using the Prism software (GraphPad Software Inc., San Diego, CA), and Excel. Statistical significance between experimental groups was assessed using an unpaired two sample Student’s t test. Data are expressed as means 6SD. A p value of less than 0.05 is considered statistically significant. Supporting Information Figure S1 MACS sorting of MLECS. Flow cytometric analysis of fractions collected during MACS sorting of lung endothelial cells from mice, after 10 weeks of HCD, showed efficient separation of CD146 positive and negative fractions. (TIF) Figure S2 Cholesterol and bodyweight of mice on HCD. Cholesterol (mg/dl) (top) and bodyweight (gr) (bottom) levels of (A) male and female p38EC-KO/ApoE2/2 and ApoE2/2 mice (p38aEC-KO/ ApoE2/2 males, n = 14; ApoE2/2 males, n = 15; p38aEC-KO/ApoE2/2 females, n = 14; ApoE2/2 females, n = 13) and (B) male and female p38aMY-KO/ApoE2/2 and ApoE2/2 mice (p38aMY-KO/ApoE2/2 males, n = 9; ApoE2/2 males, n = 15; p38aMY-KO/ApoE2/2 females, n = 8; ApoE2/2 females, n = 9), before and after 10 weeks of a HCD. Error bars represent SD. (TIF) Figure S3 Quantification of p38a expression and p38 phosphorylation in MLECs. The expression of p38a and the levels of phosphorylated total p38 in figure 4B were quantified by performing volumetric analysis of the bands identified by immunoblot with p38a and p-p38 specific antibodies. (TIF) Figure S4 Similar oxLDL uptake in p38aMY-KO/ApoE2/2 and ApoE2/2 macrophages, in vitro. (A) Quantification by flow cytometry of oxLDL uptake by thioglycolate-elicited PMs, after 50 mg/ml DiI-oxLDL stimulation for 2.5 hrs. (B) Staining of oxLDL stimulated macrophages with Oil red O (lipid staining). Foam cells, defined as cells with $10 lipid droplets, are indicated with arrows. ApoE2/2 mice, n = 3; p38aMY-KO/ApoE2/2, n = 3. (TIF) Acknowledgments We thank E. Mahlberg, T. Tropartz, J. Pfeiffer, and C. Uthoff-Hachenberg for their excellent technical assistance and Dr. Hanns-Eugen Sto¨ffler for providing us with the Radames software. We also thank the Animal Facility staff of the Institute for Genetics, University of Cologne, for excellent care of the animals used in this study. Author Contributions Conceived and designed the experiments: RK MP RG. Performed the experiments: RK. Analyzed the data: RK MP RG SX. Contributed reagents/materials/analysis tools: RK MP RG SX. Wrote the paper: RK MP. PLoS ONE | www.plosone.org 11 June 2011 | Volume 6 | Issue 6 | e21055