Fig 2: NEDD4L is a target of miR-30a-5p(A) Target prediction of the miR-30a-5p binding site on NEDD4L using TargetScan 7.2. (B) Representative western blot images and (C) quantification (n = 5) of NEDD4L in RAW264.7 macrophages transfected with miR-30a-5p or control mimics. (D) Relative luciferase analysis of RAW264.7 macrophage co-transfected with miR-30a-5p/control mimics and NEDD4L-3'UTR (WT) or NEDD4L mutated 3'UTR (MUT) with dual-luciferase assay. (E) Representative western blot images and quantification of PPAR-? (n = 4), (F) ABCA1 (n = 6), (G) ABCG1 (n = 6), (H) LDLR (n = 8), and (I) PCSK9 (n = 6) in RAW264.7 macrophages transfected with NEDD4L siRNA or control siRNA. Each dot represents an individual experiment. *p < 0.05, **p < 0.01, ***p < 0.001, ns (not significant) by unpaired t test.

Fig 3: miR-30a-5p restrained ox-LDL uptake and foam cell formation by regulating the expression of PPAR-?, ABCA1, ABCG1, LDLR, and PCSK9(A) Representative immunofluorescent stained images and (B) quantification (n = 6) of Dil-ox-LDL-treated RAW264.7 macrophages transfected with miR-30a-5p or control mimics. (C) Representative images and (D) quantification (n = 6) of oil red-stained RAW264.7 macrophages treated with ox-LDL or vehicle and transfected with miR-30a-5p or control mimics. (E) Representative transmission electron microscope images of RAW264.7 macrophages treated with ox-LDL and transfected with miR-30a-5p or control mimics. (F) Western blot images and (G) relative quantification (n = 5) of PPAR-?, ABCA1, ABCG1, LDLR, and PCSK9 in RAW264.7 macrophages transfected with miR-30a-5p or control mimics (n = 5). *p < 0.05, **p < 0.01, ***p < 0.001.

Fig 4: Smad-1/2 phosphorylation and PPAR-? ubiquitination are involved in NEDD4L-mediated M1/M2 polarization and foam cell formation(A) Representative western blot images of phosphorylation of Smad-1/2/3 in RAW264.7 macrophages treated with LPS for 15, 30, and 60 min transfected with NEDD4L or control siRNA. Representative western blot images and quantification of (B) the expression of iNOS (n = 5) and (C) CD206 (n = 5) in NEDD4L siRNA- or control siRNA-transfected RAW264.7 macrophages in the absence or presence of LPS. (D) Co-immunoprecipitation detection for total ubiquitination and (E) K48- or (F) K63- linked polyubiquitination of PPAR-? in RAW264.7 macrophages transfected with NEDD4L or control siRNA. (G) Western blot images and quantification of PPAR-? (n = 5), (H) ABCA1 (n = 5), (I) ABCG1 (n = 5), (J) LDLR (n = 5), and (K) PCSK9 (n = 5) in RAW264.7 macrophages transfected with NEDD4L or control siRNA in the absence or presence of ox-LDL. Each dot represents an individual experiment. *p < 0.05, **p < 0.01, ***p < 0.001.