Inhibition of NF-κB blocks TNFα-induced inhibition of myocardin expression and activity.

<p>VSMCs were serum-starved for 24 h and treated with TNFα (50 ng/ml). (<b>A</b>) Western blot analysis showing the effect of NF-κB inhibitor TPCK (1 µM) on TNFα-induced inhibition of myocardin and contractile marker expression. (<b>B,C</b>) Western blot and RT-qPCR analyses showing the effects of NF-κB inhibitor QNZ (1 µM) on TNFα-induced inhibition of myocardin expression and activity compared with the control (C). (<b>D,E</b>) VSMCs were treated with TNFα for different time points and the p65 subunit was stained using anti-p65 antibody and AF488-conjugated secondary antibody. The nuclear localization of the p65 subunit was determined using LSC based on AF488 florescence and propidium iodide (PI) nuclear staining. (<b>F</b>) VSMCs were treated with TNFα for different time points and cellular and nuclear proteins were extracted separately. Western blot analysis was performed to show localization of the p65 subunit in the cytoplasm and nuclei of VSMCs. qPCR results were normalized using GAPDH (n = 4, *p<0.05).</p

Model of TNFα-mediated myocardin mRNA stability.

<p>(<b>A</b>) The effect of TNFα in de-differentiated VSMCs. (<b>B</b>) The effect of TNFα in differentiated VSMCs.</p

TNFα down-regulates endogenous myocardin expression and activity.

<p>VSMCs were serum-starved for 24 h and treated with TNFα (50 ng/ml) for 24 h. (<b>A</b>) Western blot analysis showing the effects of TNFα on myocardin and contractile marker protein expression levels compared with the control (C). (<b>B</b>) RT-qPCR analysis showing the effect of TNFα on myocardin and contractile marker mRNA expression levels compared to control (C). (<b>C,D</b>) Western blot and RT-qPCR analysis showing the concentration dependent effects of TNFα on myocardin expression and activity. qPCR results were normalized using GAPDH (n = 8, *p<0.05).</p

TNFα increases proliferation of VSMCs.

<p>VSMCs were serum-starved for 24 h, treated with the indicated drugs for 24 h and labeled with BrdU for 60 min. Cells were then fixed with 80% ethanol and BrdU incorporation was detected using anti-BrdU antibody and AF488-conjugated secondary antibody using LSC. (<b>A</b>) Cells were treated with TNFα with and without QNZ. Graph shows the percentage of BrdU positive cells of the total scanned cells. (<b>B</b>) Cells were treated with TNFα and/or Dox to induce myocardin expression. Graph shows the percentage of BrdU positive cells of the total number of cells (n = 6, *p<0.05).</p

TNFα stabilizes myocardin mRNA which upregulates myocardin expression and activity in differentiated VSMCs.

<p>(<b>A</b>) VSMCs were serum-starved for 24 h and transcription was blocked using α-amanitin (50 µM). Cells were then treated with TNFα (50 ng/ml) for 1, 3, 6 and 9 h. Graph shows the effect of TNFα on myocardin mRNA degradation. (<b>B</b>) VSMCs containing the T-REx system for myocardin over-expression were treated with Dox. Western blot analysis shows the effect of Dox on myocardin and contractile protein expression levels. (<b>C</b>) RT-qPCR analysis showing the effect of myocardin over-expression on MRTF-A and B. (<b>D</b>) Light microscope images showing the effect of Dox on VSMC morphologies. (<b>E,F,G</b>) VSMCs were serum-starved for 24 h and treated with TNFα (50 ng/ml) and Dox for 24 h. Representative western blots and RT-qPCR analysis show the effect of TNFα on myocardin and contractile marker protein and mRNA expression levels compared with those control without Dox (C). (<b>H</b>) VSMCs without the T-REx system to over-express myocardin were treated with TNFα and Dox to determine non-specific effects of Dox on myocardin. RT-qPCR analysis shows the effect of TNFα and Dox on myocardin mRNA expression levels compared with the control (C). (<b>I</b>) Primary rat aortic VSMCs (passage 2) were serum-starved for 24 h and treated with TNFα (50 ng/ml) for 24 h. Representative western blots show the effect of TNFα on myocardin and contractile marker protein expression levels. (<b>J,K</b>) Western blot and RT-qPCR analyses showing the concentration- and time-dependent effects of TNFα on myocardin and contractile marker expression in VSMCs treated with Dox. qPCR results were normalized using GAPDH (n = 7, *p<0.05).</p

TNFα decreases contractility of VSMCs.

<p>VSMCs were serum-starved for 24 h, subcultured into collagen gel matrix and treated with TNFα with or without QNZ for 24 h. (<b>A</b>) Representative images of collagen gels showing the change in area of the gel. (<b>B</b>) The cross-sectional area was analyzed using ImageJ and plotted on a graph to show the changes in the collagen area (n = 3, *p<0.05).</p

NC enhances intracellular ox-LDL degradation through facilitation of lipophagy.

<p>(A) Representative photomicrographs of colocalization of lipid droplets (LDs) with LC3-II in THP-1 cells. After THP-1 cells were transfected with GFP-LC3II for 24 h, cells were treated with ox-LDL (100 μg/ml), ox-LDL (100 μg/ml) + NC (10 μM), ox-LDL (100 μg/ml) + NC (10 μM) + 3-MA (10 mM), ox-LDL (100 μg/ml) + NC (10 μM) + CQ (20 μM), and ox-LDL (100 μg/ml) + vehicle for 36 h. After washing with PBS, cells were fixed with 4% paraformaldehyde, and then stained with Nile Red (10 ng/ml) for 30 min to evaluate the accumulation of LDs. The colocalization of LDs with LC3II was examined by immunocytochemistry as described in Methods section. (B) The percentage of colocalization of LDs with LC3-II was quantified with Image J software (n = 16 cells/group). (C) Representative photomicrographs of LD accumulation in THP-1cells. Cells were incubated with ox-LDL (100 μg/ml) conjugate without or with NC of 36 h, or pre-incubated with ox-LDL for 4 h, and then treated without or with NC for additional 36 h. The intracellular LD accumulation was evaluated by Nile Red (10 ng/ml) staining. (D) Average number of LDs in THP-1 cells was quantified (n = 12 cells/group). All the data were shown as mean ± SEM of 3 independent experiments. NS: no significant difference. *<i>P</i> < 0.05, **<i>P</i> < 0.01.</p

NC reduces ox-LDL accumulation in THP-1 cells via activation of autophagy.

<p>(A) Representative photomicrographs of THP-1 cells loaded with Dil-ox-LDL. Cells were treated with ox-LDL (100 μg/ml), ox-LDL (100 μg/ml) + NC (10 μM), ox-LDL (100 μg/ml) + NC (10 μM) + 3-MA (10 mM), ox-LDL (100 μg/ml) + NC (10 μM) + CQ (20 μM), and ox-LDL (100 μg/ml) + vehicle for 36 h. After washing 3 times, cell lysates were collected for the measurement of fluorescence. Nuclei were counterstained with DAPI. (B) Quantification of fluorescence intensity from experiments as described in (A). (C) TEM was used to evaluate foam cell formation and autophagy alteration. THP-1 cells were treated with vehicle, ox-LDL (100 μg/ml), ox-LDL + NC (10 μM)), ox-LDL (100 μg/ml) + NC (10 μM) + 3-MA, ox-LDL (100 μg/ml) + NC (10 μM) + CQ (20 μM), and ox-LDL (100 μg/ml) + vehicle for 36 h, respectively. Mitochondria (M), the nucleus (N), lysosomes (L), autophagosomes (APs), autophagolysosomes (ALs), and lipid droplets (LDs) were indicated. (D, E, and F) Average number of APs, ALs, and LDs was quantified as described in Methods section (n = 12 cells/group). All the data were shown as mean ± SEM of 3 independent experiments. *<i>P</i> < 0.05, **<i>P</i> < 0.01.</p

Autophagy prevents ox-LDL-induced foam cell formation in THP-1 cells.

<p>THP-1 cells were treated with the vehicle solution (control), control + CQ (10 μM), ox-LDL (100 μg/ml), ox-LDL (100 μg/ml) + CQ (10 μM), ox-LDL (100 μg/ml) + Rap (20 μM) for 36 h, respectively. (A) LC3-I (18 kDa), LC3-II (16 kDa), and p62 (62 kDa) protein levels were detected by western blot analysis. Each lane contained 20 μg proteins for all experiments. (B) and (C) The LC3-II/LC3-I ratio and p62 level were quantified with Sigma Scan Pro5 software. Each lane was normalized to that of GAPDH (kDa). (D) Oil red O staining was used to evaluate THP-1 foam cell formation (magnification × 200). (D) Intracellular total cholesterol content was determined by enzymatic assay. All the data were shown as mean ± SEM of three independent experiments. *<i>P</i> < 0.05, **<i>P</i> < 0.01.</p

NC promotes cholesterol efflux via restoring autophagy flux.

<p>(A)and (B) THP-1 cells were incubated in medium containing 100 μg/ml ox-LDL that was labeled with 0.5 μ Ci/mL ³H-cholesterol (PerkinElmer) for an additional 30 h and then treated with vehicle, NC (10 μM), NC (10 μM) +3-MA (10 mM), and NC (10 μM) + CQ (20 μM) for additional 6 h. Subsequently, ApoA1- or HDL-mediated cholesterol efflux was analyzed by liquid scintillation counting assay. The efflux is expressed as the percentage of effluxed ³H-cholesterol/total cell cholesterol ³H-cholesterol content (effluxed ³H-cholesterol + intracellular ³H-cholesterol) × 100%. All the data were shown as mean ± SEM of 3 independent experiments. NS: no significant difference. *<i>P</i> < 0.05, **<i>P</i> < 0.01.</p