Fig 2: MSC-TGF-ß1 altered the phenotypes of mice peritoneal macrophages. Macrophages were harvested from mice peritoneal cavity and analysed by flow cytometry on their expression of certain makers. A, CD206 expression in macrophages at day 14 after transplantation B, CD206 expression in macrophages at day 21 after transplantation. C, iNOS expression in macrophages at day 14 after transplantation. D, Quantitative analysis of the expression of CD206 in macrophages at day 14 after transplantation. E, Quantitative analysis of the expression of CD206 in macrophages at day 21 after transplantation. F, Quantitative analysis of the expression of iNOS in macrophages at day 21 after transplantation. Data are representative of three independent experiments with 5-7 mice per group. Data are presented as the mean ± SD. ***P < .001

Fig 3: miR-30a-5p overexpression decreased the m1 macrophage polarization and increased M2 macrophage polarization in vivo(A) Representative immunofluorescent stained images and (B) quantification (n = 5) of the FISH assay for expression of miR-30a-5p in the aortic root of mice with different treatments. Representative immunofluorescent staining images and quantification (n = 5) of CD31 (ECs, C and D), a-SMA (VSMCs, E and F), MOMA-2 (macrophage, G and H), iNOS (M1 macrophage, I and J), and CD206 (M2 macrophage, K and L) in the plaque of aortic root in mice with different treatments. *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.

Fig 5: Macrophage polarization is dependent on SOCE channels, and inhibition of these channels disrupts cytokine release(A and B) Cytokine analysis by colorimetric analysis in supernatants of M1 and M2 activated bone marrow-derived macrophage (BMDM) from wild type after treatment with and without SKF96365 (10 µM for 6 h). Data shown are representative of three independent experiments performed in triplicates. Bar graphs depict average ± SD for relative values, ***p = 0.001 (Student's t test).(C and D) Flow cytometer analysis on M1 and M2 activated wild-type mice BMDM with and without SKF96365 (10 µM for 6 h) to show how Ca2+ inhibition interrupts macrophage polarization by blocking surface (CD80 + CD206+) and internal (iNOS and Arginase-1) markers for the different phenotypes. Data shown are representative of three independent experiments.(E) Immunoblot analysis showing the level of expression of wild-type mice M1 activated BMDM pathway genes pNF-?B, NF-?B, pJNK, JNK in media treated for 6 h with SKF96365 (10 µM). Data shown are representative of three independent experiments with similar results.(F) Immunoblot analysis showing the level of expression of wild-type mice M2 activated BMDM pathway genes Arg1, pSTAT6 in media treated for 6 h with SKF96365 (10 µM). Data shown are representative of three independent experiments.(G–I) Primary macrophages derived from bone marrow were pulsed with medium alone (NS), IFN? at 20 ng/mL, IL4 at 20 ng/mL, and cultured for a total of 2, 6, and 24-h period. Cells were harvested and chromatin immunoprecipitation (ChIP) experiments was performed with anti-pNF-?B, anti-pSTAT1, or anti-pSTAT6 at the promoter regions of TRPC1 was assessed by qPCR and expressed as percentage of input. The mean ± SE of NF-?B (G), pSTAT1 (H), pSTAT6 (I) are shown.(J–L) Time-dependent binding to Orai1 promoter region by pSTAT3, pPPARg, and pIRF4 upon IL-4 stimulation; data presented are mean ± SE for each condition. Significant differences were measured by Student's t test (*p < 0.05; NS, non-significant, n = 3).(M–O) Immunoblot analysis showing the level of expression of phosphorylated STAT1 and STAT6 in M1 and M2 cells that were treated for 24 h in 0.5 or 2 mM external calcium. Data shown are representative of three independent experiments. Representative confocal images showing localization of pSTAT1(N) and pSTAT6(O) in cytoplasm and in the nucleus (co-localization with DAPi staining) in cells treated with various calcium concentrations. Scale bar, 10 µm.