Ox-LDL triggers NF-kB and MAPK signaling.

<p>(<b>A</b>) – Western blots for phospho-IκBα and p44/42 MAPK with corresponding graphs depicting relative densities of bands normalized for β-actin in relation to control (fold change); (<b>B</b>) – Immunostaining for phosphor-p65 (NFκB) in MCF10A. Note that cells exposed to ox-LDL display translocation of protein to the nucleus (arrows) within 30 minutes of exposure and that percentage of cells with translocated p65 increases with time. (<b>C</b>) - Western blots for phospho-p65 and phosphor-p44/42 MAPK within short (30 min) exposure to 20 µg/ml ox-LDL with corresponding graphs depicting relative densities of bands normalized for β-actin in relation to control (fold change); (*) – Significant difference (p<0.05) compared to control.</p

Ox-LDL increases expression of miR-21 accompanied with PI3K/Akt upregulation and inhibition of target genes.

<p>(<b>A</b>) – qPCR analysis for expression of hsa-miR-21; (<b>B</b>) – qPCR analysis for expression of hsa-miR-21 target genes in control (white columns) and ox-LDL treated (20 µg/ml, 12 hours, black columns) cells; (<b>C</b>) – Western blot for PTEN, Akt and PI3K; (<b>D</b>) - qPCR analysis for expression of PI3K, Akt and PDCD4 genes in control cells and miR-21 inhibitor transfected cells treated with ox-LDL; (<b>E</b>) – Western blots for phospho-Akt within short (30 min) exposure to 20 µg/ml ox-LDL with corresponding graph depicting relative densities of bands normalized for β-actin in relation to control (fold change); (*) – significant difference compared to control. (†) - Significant difference (p<0.05) compared to ox-LDL treated non-transfected cells.</p

Ox-LDL exposure is followed by upregulation of NADPH oxidase, LDL modifying enzymes and SOD1.

<p>(<b>A</b>) – Western blots for Lipoxygenases-12, lipoxygenase -15-2 and P22^phox and P47^phox subunits of NADPH oxidase (treatment with 20 µg/ml ox-LDL for 2 and 12 hours); (<b>B</b>) – corresponding graphs depicting relative densities of bands normalized for β-actin in relation to control (fold change); (<b>C</b>) – Western blots for SOD1 and SOD2 (similar conditions) and (<b>D</b>) – corresponding graphs depicting relative densities of bands normalized for β-actin in relation to control (fold change). (*) – Significant difference (p<0.05) compared to control.</p

Ox-LDL stimulates cell proliferation and expression of scavenger receptors.

<p>(<b>A</b>) – For evaluation of proliferative response to ox-LDL measured by cell count and MTT assay, MCF10A cells were exposed to different concentrations of ox-LDL (1–100 µg/ml) for 24 hours; (<b>B</b>) – uptake of Dyl-ox-LDL by MCF10 A cells was visualized by fluorescent microscopy after 2 hours of incubation with 1 µg/ml Dil-ox-LDL; (<b>C</b>) – Western blot for scavenger receptors and (<b>D</b>) – graph depicting relative densities of bands normalized for β-actin in relation to control (fold change). (*) – significant difference (p<0.05) compared to control. For these experiments, MCF10A cells were treated with 20 µg/ml ox-LDL for 2 and 12 hours.</p

Senescence-dependent decline in <i>VCAM-1</i> and <i>ICAM-1</i> expression.

<p>A. Expression of <i>VCAM-1</i> and <i>ICAM-1</i> in P4, P8 and P12 cells evaluated by qPCR; B. Protein content for <i>VCAM-1</i> and <i>ICAM-1</i> evaluated by Western blots. Data from 3 independent experiments are expressed as fold change in relation to P4 values ± SEM. (*) – p<0.05 compared to P4.</p

The expression of <i>LOX-1</i> in senescent endothelial cells.

<p><b>A. Changes in </b><b><i>LOX-1</i></b><b> expression (qPCR, left) and content (right) with senescence.</b> Data are expressed as fold change in relation to P4 values; data in mean ± SEM from 3 independent experiments. (*) – p<0.05 compared to P4. B. Representative images of Dil-ox-LDL uptake by P4 and P12 endothelial cells. C. LOX-1 immunostaining of aortas from 5- and 50-wk old mice (data representative of 3 separate aortas). Note that the LOX-1 signal in endothelial cells in 50-wk old mice aortas is almost undetectable.</p

Increased susceptibility to apoptosis in late passage HUVECs.

<p>A. Expression of <i>BAX</i> and <i>BCL2</i> mRNA in P4, P8 and P12 cells evaluated by qPCR; B. <i>BAX</i> and <i>BCL2</i> protein content evaluated by Western blots. Data from 3–5 independent experiments are expressed as fold change in relation to P4 values ± SEM. C. Apoptosis in HUVECs in response to 24-hr exposure to TNFα (50 µg/ml) measured using polycaspase staining. Upper panel- Representative images of apoptotic cells (green) in P4 and P12 cells treated with TNFα. Lower panel – Quantitation of apoptotic response as a percentage of total cell number. (*) – p<0.05 compared to P4; (†) – p<0.05 compared to the same passage control.</p

qPCR primers used in this study.

<p>qPCR primers used in this study.</p

Senescence affects morphology and angiogenic potential of endothelial cells.

<p>A. Cell size in late (P12) passage cells is increased by 36% compared to early (P4) passage cells as judged by a number of cells within the field of view in confluent cultures, and the number of nuclei stained with DAPI in 100% confluent cultures is smaller in P12 cells. B. Graph depicts the length of tubes formed by P4, P8 and P12 cells on matrigel in the absence or the presence of 20 ng/ml VEGF. Values are expressed as fold change in relation to unexposed P4 Control. C. Representative Western blots for 4-HNE modified proteins in lysates from P4 and P12 HUVECs. D. Representative Western blots for VE-cadherin and von Willebrand factor, and E. Relative protein content in relation to P4 values; data in mean ± SEM from 3 independent experiments. (*) – p<0.05 compared to P4 cells; (†) – p<0.05 compared to the same passage Control.</p

Senescence-dependent changes in sub-cellular localization of NF-kB p65.

<p>A. Changes in NF-kB p65 and <i>IκBα</i> content in P4 and P12 cells evaluated by Western blots. Data are expressed as fold change in relation to P4 values ± SEM. B. NF-kB p65 immunostaining of P4 and P12 cells. Note that the significant fraction of p65 translocates to the nuclei in P12 cells.</p