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• Figure I - (TIF) (3.59 MB). Negative controls for immunohistochemistry. Negative controls for GRP78 and TDAG51 (addition of anti-goat secondary antibody) (a), CHOP (heat retrieval, Triton X, addition of anti-rabbit secondary antibody) (b), and Mac-3 (heat retrieval, addition of anti-rat secondary antibody) (c) are indicated.
• Figure II - (TIF) (5.28 MB). Morphometric and immunohistochemical analysis of an early atherosclerotic lesion. Consecutive sections of an early atherosclerotic lesion from apoE-/- mice were stained with hematoxylin and eosin (H&E in a), or immunostained for macrophages with Mac-3 antibody (b), endothelial cells with an anti-von Willebrand factor antibody (c), GRP78 (d) or phospho-PERK (e). Bar =50 μm.
• Figure III - (TIF) (10.8 MB). Identification of calreticulin and PDI in early and late atherosclerotic lesions from apoE-/- mice. Intimal macrophages (arrow in a) and macrophage foam cells in fatty streaks (b) stained positively for calreticulin. The majority of cells in the cap region of advanced lesions were positive for calreticulin (c). In some fatty streaks and advanced lesions, SMC in the media were also immunopositive for calreticulin (b and c). PDI immunostaining was similar to that of GRP78 (d to f) except that PDI staining was observed in medial SMC underlying the early lesions. L, lumen. Bar=50 μm.
• Figure IV - (TIF) (7.77 MB). Apoptosis in advanced lesions. Consecutive sections stained for TUNEL (a), cleaved caspase-3 (b) and CHOP (c). Colocalization of TUNEL and cleaved caspase-3 was found in some necrotic areas (arrowheads in a and b), whereas other areas with nuclear fragmentation and positive for TUNEL labeled poorly for cleaved caspase-3 (arrows in a and b). Some colocalization between TUNEL and CHOP staining was observed in some lesional macrophages overlying the necrotic core (arrows in a and c).
• Figure V - (TIF) (3.82 MB). Positive and negative controls for apoptotic cell death on mouse thymus tissue. Thymocytes with apoptotic nuclear morphology were positive for TUNEL (a) and negative when dTT enzyme was omitted (b). Cleaved caspase-3 labeled cytoplasm of thymocytes with condensed nuclei (c), while omission of the primary antibody resulted in no staining (d). TUNEL and cleaved caspase-3 co-localization by fluorescence (TUNEL; nuclei red, cleaved-caspase-3; cytoplasm green, arrows in e).
• Figure VI - (TIF) (1.11 MB). Differentiation of human blood peripheral monocytes into macrophages activates the UPR and increases the expression of GRP78, GRP94, CHOP and XBP-1. Human blood peripheral monocytes were treated with 50 ng/mL macrophage colony stimulating factor (MCSF) to induce macrophage differentiation. Panel a, electron micrograph of a human blood peripheral monocyte. Panel b, electron micrograph of a differentiated macrophage following treatment with MCSF for 7 days. Panel c, expression of GRP78, GRP94 CHOP and XBP-1 in differentiating macrophages. Human peripheral blood monocytes were treated with MCSF for time periods up to 7 days and total cell lysates were assessed by immunoblot analysis. Results indicate a time-dependent increase in the expression of GRP78, GRP94, CHOP and XBP-1 following differentiation of monocytes to macrophages.