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(A) PACERR expression in total RNA isolated from human carotid endarterectomy samples where advanced/central plaques are separated from peripheral plaque and sequencing was done on both. (B) scRNA-seq of carotid plaque samples. (C) THP-1 cells differentiated into macrophages using 10 nM PMA for 48–72 h then treated with ApoA1 (50µg/ml), HDL (50µg/ml), acLDL (37.5µg/ml), oxLDL (10µg/ml), or LPS (100ng/ml) for 24 hours followed by qPCR for gene expression analysis. (D) THP-1 macrophages were treated with 50µg/ml HDL, <t>HDL2</t> or <t>HDL3</t> for 24 hours and qPCR for gene expression was used, western blot to measure COX-2 protein levels and mass spectrometry was used to measure 6k-PGF1a/PGI2 and PGE2 in the cell media. (E) THP-1 macrophages were pre-treated with 5µM celecoxib for 1 hour before addition of 50µg/ml HDL or 100ng/ml LPS for 24 hours. (F) THP-1 macrophages were pre-treated with 5µM Bay-117082 for 1 hour before addition of 50µg/ml HDL or 100ng/ml LPS for 24 hours. (G) THP-1 macrophages stably expressing CREB shRNA were treated with 50 µg/ml or 100ng/ml LPS for 24 hours then qPCR was used to measure gene expression. Values are mean ±SEM of three independent experiments. *p<0.05, **p<0.01, ***p<0.005, ****p<0.001 versus untreated.
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(A) PACERR expression in total RNA isolated from human carotid endarterectomy samples where advanced/central plaques are separated from peripheral plaque and sequencing was done on both. (B) scRNA-seq of carotid plaque samples. (C) THP-1 cells differentiated into macrophages using 10 nM PMA for 48–72 h then treated with ApoA1 (50µg/ml), HDL (50µg/ml), acLDL (37.5µg/ml), oxLDL (10µg/ml), or LPS (100ng/ml) for 24 hours followed by qPCR for gene expression analysis. (D) THP-1 macrophages were treated with 50µg/ml HDL, HDL2 or HDL3 for 24 hours and qPCR for gene expression was used, western blot to measure COX-2 protein levels and mass spectrometry was used to measure 6k-PGF1a/PGI2 and PGE2 in the cell media. (E) THP-1 macrophages were pre-treated with 5µM celecoxib for 1 hour before addition of 50µg/ml HDL or 100ng/ml LPS for 24 hours. (F) THP-1 macrophages were pre-treated with 5µM Bay-117082 for 1 hour before addition of 50µg/ml HDL or 100ng/ml LPS for 24 hours. (G) THP-1 macrophages stably expressing CREB shRNA were treated with 50 µg/ml or 100ng/ml LPS for 24 hours then qPCR was used to measure gene expression. Values are mean ±SEM of three independent experiments. *p<0.05, **p<0.01, ***p<0.005, ****p<0.001 versus untreated.

Journal: bioRxiv

Article Title: The Regulation of COX-2, ABCA1 and ABCG1 by the lncRNA PACERR links the inflammatory response and cholesterol homeostasis

doi: 10.1101/2025.06.02.657432

Figure Lengend Snippet: (A) PACERR expression in total RNA isolated from human carotid endarterectomy samples where advanced/central plaques are separated from peripheral plaque and sequencing was done on both. (B) scRNA-seq of carotid plaque samples. (C) THP-1 cells differentiated into macrophages using 10 nM PMA for 48–72 h then treated with ApoA1 (50µg/ml), HDL (50µg/ml), acLDL (37.5µg/ml), oxLDL (10µg/ml), or LPS (100ng/ml) for 24 hours followed by qPCR for gene expression analysis. (D) THP-1 macrophages were treated with 50µg/ml HDL, HDL2 or HDL3 for 24 hours and qPCR for gene expression was used, western blot to measure COX-2 protein levels and mass spectrometry was used to measure 6k-PGF1a/PGI2 and PGE2 in the cell media. (E) THP-1 macrophages were pre-treated with 5µM celecoxib for 1 hour before addition of 50µg/ml HDL or 100ng/ml LPS for 24 hours. (F) THP-1 macrophages were pre-treated with 5µM Bay-117082 for 1 hour before addition of 50µg/ml HDL or 100ng/ml LPS for 24 hours. (G) THP-1 macrophages stably expressing CREB shRNA were treated with 50 µg/ml or 100ng/ml LPS for 24 hours then qPCR was used to measure gene expression. Values are mean ±SEM of three independent experiments. *p<0.05, **p<0.01, ***p<0.005, ****p<0.001 versus untreated.

Article Snippet: High density lipoproteins (HDL, HDL2 and HDL3) were purchased from Lee biosolutions (cat # 361-10-0.1) or academy biomedical solutions (80P-HD-101, 80P-HD2-101, 80P-HD3-101) and used at 50µg/ml, acetylated LDL (acLDL) was purchased from Kalen Biomedical, LLC (770201-6) and used at 37.5µg/ml.

Techniques: Expressing, Isolation, Sequencing, Gene Expression, Western Blot, Mass Spectrometry, Stable Transfection, shRNA