Fig 2: Superlow-dose LPS induces inflammatory polarization of neutrophils in vitro.Neutrophils were purified from the BM of wild-type C57BL/6 mice and treated with PBS, superlow-dose LPS (100 pg/ml), and/or oxLDL (10 µg/ml) for 2 days. (A) Levels of MMP9, LTB4, and MPO were determined by ELISA (n = 3 for each group). (B) The surface phenotype of neutrophils was analyzed by flow cytometry (n = 3 for each group). (C) Levels of miR-24 and miR-126 were determined by real-time reverse transcription polymerase chain reaction (RT-PCR) (n = 4 for each group). (D) The expressions of oxCaMKII and 5-LOX were determined by Western blot. (E) Representative histogram and quantification of p-STAT1 level as determined by flow cytometry (n = 3 for each group). (F) Representative histograms and quantification of ATF4 and KLF2 levels as determined by flow cytometry (n = 5 for each group). Data are representative of three independent experiments, and error bars represent means ± SEM. *P < 0.05, **P < 0.01, and ***P < 0.001, (A to C and E) one-way analysis of variance (ANOVA) and (F) Student’s t test.

Fig 3: Immunolocalization of Enzyme Positive Cells at 4 days after Fracture.The top image shows a fractured mouse femur stained with safranin-O (orange) and counter stained with fast green (green) and hematoxylin (black; CB: cortical bone; FX: fracture site; scale bar: 300 um). Bottom images show immunohistochemical staining of different cell types with rabbit IgG (Neg.) or with antibodies to COX-1, COX-2, 5-LO, or LTA4H (brown; scale bar: 50 um). Immunohistochemistry specimens were counter stained with methyl green. The higher magnification images are labeled with the primary antibody target (COX-1, COX-2, 5-LO, LTA4H, or Neg.) and with a letter indicating cell type and location as listed in Figure 1.

Fig 4: Quantitative Analysis of Fracture Site COX-2 Positive Osteoclasts and 5-LO Positive Fibroblasts.The number of external callus osteoclast (triangles), internal callus osteoclast (inverted triangles), and callus fibroblasts (circles) were counted (right axis). The percentage of osteoclast that also were positive for COX-2 by immunohistochemical detection were calculated for the external (right bars)and internal callus (left bars) at each time point and are shown as mean values (+ S.E.M.; Panel A; left axis). The percentage of fibroblasts positive for 5-LO by immunohistochemistry were calculated at each time point and are shown as mean values (+S.E.M.; Panel B; left axis). Note that the X-axis scales (Days after Fracture) are not linear with respect to time.

Fig 5: Impaired binding of TS-5-LOW13/W75/W102A with the SPT complex. TS-5-LO and TS-5-LOW13/W75/W102A were pulled down from Ag-activated BMMCL, size-fractionated by SDS-PAGE, and analyzed by immunoblotting with corresponding antibodies (a-). The input of the analyzed proteins is also shown. Data are representative of three independent experiments.