MCC950 compound abrogates oxLDL-induced increase in pro-IL-1β protein expression.

THP-1 cells were exposed to oxLDL, the NLRP3 inhibitor MCC950 (MCC) or their combination (MCC+OxL). Cell lysates were subjected to SDS-PAGE and immunoblotted with anti-pro-IL-1β (upper panel) or anti-vinculin antibody (bottom panel), as indicated. The band densities, relative to vinculin (loading control), were quantified using ImageJ (NIH, Bethesda, MD). Veh, vehicle. N = 3/group, * P (TIF)</p

Olaparib attenuates the oxLDL-mediated increase in pro-IL-1β and pro-IL-18 protein expression.

THP-1 monocytes were exposed to oxLDL, the PARP inhibitor olaparib (Olapa), or the NLRP3 inhibitor MCC950 (MCC), individually or in combination as indicated. Cell lysates were subjected to SDS-PAGE and immunoblotted with anti-pro-IL-1β (top), anti-pro-IL-18 (middle) or anti-vinculin antibody (bottom, for loading control). The densities of pro-IL-1β, pro-IL-18 over vinculin were quantified by densitometry and normalized to vehicle. The band densities, relative to vinculin, were quantified using ImageJ (NIH, Bethesda, MD). N = 4/group, *P<0.05 vs vehicle, #P<0.05 vs oxLDL only, one-way ANOVA, Newman-Keuls test.</p

Olaparib inhibits oxLDL-induced NIK protein expression and p100 phosphorylation.

The THP-1 monocytes were exposed to oxLDL, the PARP inhibitor olaparib (Olapa), or the NLRP3 inhibitor MCC950 (MCC), as indicated. The cell lysates were subjected to SDS-PAGE and immunoblotted with anti-NIK (top), anti-phospho-p100 (middle) or anti-vinculin antibody (bottom, for loading control), as indicated. The band densities, relative to vinculin, were quantified using ImageJ (NIH, Bethesda, MD). N = 4/group, *P<0.05 vs vehicle, #P<0.05 vs oxLDL only, one-way ANOVA, Newman-Keuls test.</p

Olaparib inhibits oxLDL-enhanced mitochondrial ROS production.

THP-1 monocytes were exposed to oxLDL, the PARP inhibitor olaparib (Olapa), or the NLRP3 inhibitor MCC950 (MCC), individually or in combination, as indicated. Mitochondrial ROS production was measured by MitoSox Red fluorescence intensity using flow cytometry. Median and SD values are summarized from three independent experiments (n = 3/group). *P<0.05 vs vehicle, #P<0.05 vs oxLDL only, one-way ANOVA, Newman-Keuls test.</p

Olaparib inhibits oxLDL-induced increase in NF-κB activity.

The THP-1 monocytes were exposed to oxLDL, the PARP inhibitor olaparib (Olapa), or the NLRP3 inhibitor MCC950 (MCC) as indicated. The cell lysates were subjected to SDS-PAGE and immunoblotted with anti-IKK-α (top), anti-phospho-IκBα (middle) or anti-vinculin antibody (bottom, for loading control), as indicated. The band densities, relative to vinculin, were quantified using ImageJ (NIH, Bethesda, MD). N = 4/group, *P<0.05 vs vehicle, #P<0.05 vs oxLDL only, one-way ANOVA, Newman-Keuls test.</p

Olaparib inhibits oxLDL-mediated NLRP3 inflammasomes activity.

THP-1 monocytic cells were exposed to oxLDL, the PARP inhibitor olaparib (Olapa), or the NLRP3 inhibitor MCC950 (MCC), individually or in combination as indicated. Proteins in cell lysates were detected with anti-NLRP3, anti-ASC, and anti-pro-caspase-1 (pro-CASP1) antibodies, as indicated. The band densities, relative to vinculin (loading control) were quantified using ImageJ (NIH, Bethesda, MD). N = 4/group, * P#P<0.05 vs oxLDL only, one-way ANOVA, Newman-Keuls test.</p

Olaparib inhibits oxLDL-induced monocyte adhesion and foam cell formation.

(A) THP-1 monocytes were exposed to oxLDL, the PARP inhibitor olaparib (Olapa), or the NLRP3 inhibitor MCC950 (MCC) as indicated. The monocytes attached to human umbilical vein endothelial cells (HUVECs) were counted using an inverted light microscope and analyzed by ImageJ software (NIH, Bethesda). Data are from four independent experiments (n = 4/group). *P<0.05 vs vehicle, #P<0.05 vs oxLDL only, one-way ANOVA, Newman-Keuls test. (B) THP-1 cells were exposed to PMA followed by compounds as in (A) as described in Methods section. Formation of foam cells was assayed by oil red-O staining and imaged by an inverted light microscope. ORO, oil red-O staining. Data are from three independent experiments (n = 3/group). Median and SD values are shown. *P<0.05 vs vehicle, #P<0.05 vs oxLDL only, one-way ANOVA, Newman-Keuls test.</p

Olaparib inhibits OxLDL-mediated increase in IL-1β and IL-18 secretion.

THP-1 cells were exposed to OxLDL, the PARP inhibitor olaparib (Olapa), or the NLRP3 inhibitor MCC950 (MCC). Secreted IL-1β (A) and IL-18 (B) in the cell culture medium were quantified by ELISA. N = 3/group, * P (TIF)</p

Exposure to oxLDL increases pro-IL-1β protein expression in a concentration- and time-dependent manner.

(A) THP-1 cells were exposed to the indicated concentrations of oxLDL for 24 hr. (B) THP-1 cells were exposed to 100 μg/mL oxLDL at the indicated time points. The band densities of pro-IL-1β and vinculin bands (the latter as a loading control), were normalized with the bands in the vehicle-treated cells (0 μg/mL) from three independent experiments (n = 3/group), representing a total of seven replicates. *P<0.05 vs 0 hr, one-way ANOVA, Newman-Keuls test.</p

Olaparib suppresses oxLDL-enhanced NF-κB target-gene VCAM-1 protein expression in THP-1 monocytes.

The cells were exposed to oxLDL, the PARP inhibitor olaparib (Olapa), or the NLRP3 inhibitor MCC950 (MCC), as indicated. The cell lysates were subjected to SDS-PAGE and immunoblotted with anti-VCAM-1 (upper panel) or anti-vinculin antibody (lower panel). The band densities, relative to vinculin (loading control), were quantified using ImageJ (NIH, Bethesda, MD). N = 4/group, * P<0.05 vs vehicle, #P<0.05 vs oxLDL only, one-way ANOVA, Newman-Keuls test. VCAM-1, vascular cell adhesion molecule 1.</p