Fig 2: Siglec-E downregulates CD36-mediated signaling. a WT peritoneal macrophages were treated with oxLDL (50 µg/ml) for indicated times. Cell lysates were prepared, followed by immunoprecipitation with anti-Siglec E antibody. The immunoprecipitates were then subjected to Western blot analysis with indicated antibodies and analyzed by densitometry. The value at zero time point was referred to 1. The quantitative data are mean ± SE of 3 independent experiments. *P < 0.05 vs zero time point. b WT and SE-/- macrophages were treated with oxLDL (50 µg/ml) for indicated times, and the cell lysates were analyzed by Western blotting with anti-phosphoVAV and VAV antibodies and analyzed by densitometry. The value at zero time point was referred to 1. The quantitative data are mean ± SE of 3 independent experiments. *P < 0.05 vs WT group at the same time point. c The cell lysates prepared from WT and SE-/- macrophages treated with oxLDL for indicated times as described above were subjected to immunoprecipitation with anti-VAV antibody. The immunoprecipitates were subjected to Western blot analysis with indicated antibodies and analyzed by densitometry. The value at zero time point was referred to 1. The quantitative data showing relative SHP-1/VAV are mean ± SE of 3 independent experiments. *P < 0.05 vs WT group at the same time point. d WT and SE-/- macrophages were pretreated with 10 µM of NSC87877 for 30 min in culture, followed by incubation with Dil-oxLDL (10 µg/ml) for another 1 h. The accumulation of intracellular Dil-oxLDL was examined by confocal microscopy. e The quantitative results of (d). Data shown are the mean ± SE of 3 independent experiments. *P < 0.05 vs untreated WT cells; IP < 0.05 vs untreated WT cells

Fig 3: Correlation between Vav1, miR-29b and Akt2 in MDA-MB-231-derived xenografts. (A) Representative immunochemical analysis of MDA-MB-231 cells stably expressing an empty vector or Vav1. (B) Representative immunohistochemical analysis with the anti-Akt2 antibody of xenografts from the above-described MDA-MB-231 cells. (C) Analysis of Akt2 staining in xenografts that reports the Intensity of Positive pixels/Area (Ip/µm2). (D) qRT-PCR analysis (2−ΔCt) of miR-29b-3p in laser micro-dissected xenografts sections. * p < 0.05 between bars.

Fig 4: Correlation between miR-29b and Akt2 in cell lines from triple negative PDXs. (A) Amounts of miR-29b and Akt2 in PDX-derived cell lines in which Vav1 was silenced or over-expressed. The values are relative to those of cells transfected with control siRNAs or empty vectors (Ctrl), respectively, taken as 1. * p <0.05. (B) Representative Western blot analysis of PDX-derived cell lines in which miR-29b-3p was down- (Inhibitor) or up-modulated (Mimic). In (C), amounts of Akt2 as deduced from the chemiluminescence signal normalized with β-Tubulin. * p < 0.05 compared to transfection with scramble oligonucleotides (NC). All the data are the means of 3 separate experiments ± SD.

Fig 5: Correlation between Vav1, miR-29b and Akt2 in MDA-MB-231 cells. (A) qRT-PCR analysis of miR-29b-3p and miR-615-3p in MDA-MB-231 cells in which Vav1 was down-modulated or over-expressed. The values were determined using the 2 −ΔΔCT method and the asterisks indicate statistically significant differences compared to respective controls (Ctrl) taken as 1. (B) Representative immunoblot analysis of Akt2 in lysates from MDA-MB-231 cells transfected with miR-29b-3p inhibitor or mimic. In (C), amounts of Akt2 as deduced from the chemiluminescence signals normalized with β-Tubulin. The asterisks indicate significant differences compared to transfection with scramble oligonucleotides (Negative control, NC), taken as 1. All the data represent the mean of three separate experiments ± SD. * p < 0.05.