Fig. 1: Identification of the lncRNA MAARS in lesional intima.

a RNA derived from aortic intima of LDLR^−/− mice (n = 3; each sample represents RNA pooled from two mice) that were placed on an HCD for 0 weeks (group 1), 2 weeks (group 2), 12 weeks (group 3), and 18 weeks after 6 weeks of resumption of a normal chow diet (group 4). b RNA-Seq results for 11 lncRNA hits obtained by DESeq2/NOR analysis and expressed as fold change compared to group 1 (n = 3). c Heatmap for 11 lncRNAs that were dynamically regulated with progression and regression of atherosclerosis and expression of macrophage markers Mac2, Mac3, and F4/80, as compared to group 1 (n = 3). d RT-qPCR expression analysis for MAARS in different cell types (n = 3). e MAARS expression kinetics in macrophages differentiated from bone marrow (n = 3). f MAARS expression in body organs and PBMCs of 24 weeks old C57BL/6 mice (n = 4, left panel). g MAARS expression in C57BL6 spleen fractions isolated with magnetic beads specific for different cell types (n = 3): F4/80 (macrophages), CD8a (T cells), CD11c (dendritic cells), CD19 (B cells), and negative selection for B cells. h MAARS expression in body organs and PBMCs of 24 weeks old LDLR^−/− mice fed with high cholesterol diet (HCD) for 12 weeks (n = 5). i MAARS expression in 50,000 cells FACS sorted for F4/80- and CD11b-negative or double-positive cells isolated from aorta of ApoE^−/− mice fed HCD for 12 weeks (progression) and 18 weeks after 6 weeks of resumption to a normal chow diet (regression) (n = 5). j RNA-in situ hybridization for MAARS-probe (red), Mac-2 (green), and DAPI (blue) staining in the aortic lesions of LDLR^−/− fed HCD for 12 weeks. k Coding potential assessing tool (CPAT) predicts very low coding potential for MAARS lncRNA. l To test the coding potential, MAARS sequence was cloned upstream of 3xFlag-Tag cassette, transfected in 293 T cells, and immunoblotted for Flag antibody. Positive control was provided with the kit (n = 3 experiments). m RT-qPCR analysis for RNA derived from BMDMs separated into cytoplasmic and nuclear fractions and normalized to the cytoplasmic fraction (n = 3). n RNA-in situ hybridization for negative control- and MAARS-probes on PFA-fixed BMDMs. For all panels, values are mean ± SD; *p < 0.05; **p < 0.01; ***p < 0.001.