Skip to main content
. 2019 Jan 10;9:3118. doi: 10.3389/fimmu.2018.03118

Figure 1.

Figure 1

Myo1F enhances intercellular adhesion integrin-ανβ3-mediated in macrophages. (A) Representative immunofluorescence staining of E-cadherin (green) and F4/80 (red) in the colonic mucosa of C57BL/6J mice that were induced to colitis. Macrophages in colonic epithelium were identified as F4/80+ and epithelial cells are defined by the expression of E-cadherin. Colitis was induced by administration of 2.5% DSS in drinking water. Control animals received water alone. Nuclei were stained with Dapi (blue). Bar = 50 μm. White arrows defined areas with macrophages. Amplified images of the areas marked by a white square are shown. Bar = 30 μm. n = 10. (B) Monocytes (Mo) from bone marrow obtained from C57BL/6J mice were differentiated to macrophages (Mφ) as describe in materials and methods. Myo1F was analyzed by western blot. GAPDH was used as loading control. n = 3. Densitometric analyses obtained from those results are shown as graphs. ***p = 0.0005. (C) Myo1F was analyzed by western blot in cell lysates of BMM obtained from C57BL/6J mice that were differentiated into M0, M1, and M2 phenotype. GAPDH was used as loading control. Densitometric analyses obtained from those results are shown as graphs. *p = 0.05, **p = 0.01. Immunofluorescence staining for Myo1F (Green) was carried out in M1 macrophages obtained from C57BL/6J. n = 3. Membrane bound Myo1F was evaluated in BMM differentiated to M0, M1, and M2. Cytosolic and membrane fractions were prepared as previously reported (36). Integrin β3 marks membrane fraction and GAPDH marks cytosolic fraction. (D) Western blot for Myo1F and GFP was performed on cell lysates of RAW264.7 and J774 cells overexpressing Myo1F-GFP or GFP. Macrophages were transduced as described in material and methods. GAPDH was used as loading control. LE = Low exposure. HE = High Exposure. n = 5. (E) Cellular distribution of Myo1F was evaluated by confocal microscopy in transduced macrophages expressing Myo1F-GFP or control GFP. GFP = green. Actin = red. Nuclei = blue. Bar = 10 μm. n = 5. (F) Bright field images of RAW264.7 and J774 cells overexpressing Myo1F-GFP or GFP plated in flat surface. Bar = 20 μm. n = 5. (G) Cell adhesion assay was carried out with RAW264.7 overexpressing Myo1F-GFP or GFP. Bright field shows representative images of the assay. Cells were maintained in suspension for 30 min in serum free media in mild agitation. Bar = 20 μm. n = 5. Graphical representation of the cell aggregation is shown. (H) Western blot for αν and integrin-β3 was performed on cell lysates of RAW264.7 and J774 cells overexpressing Myo1F-GFP or GFP. GAPDH was used as loading control. Densitometric analyses obtained from those results are shown as graphs. **p = 0.01, ***p = 0.0005. (I) Cell adhesion assay was carried out with RAW264.7 overexpressing Myo1F-GFP or GFP in presence of cilentigide or vitronectin. Bright field shows representative images of the assay. Bar = 10 μm. n = 3. (J) Cell adhesion assay was carried out with RAW264.7 overexpressing Myo1F-GFP or GFP in presence of cilentigide or vitronectin. Bright field shows representative images of the assay. Bar = 10 μm. n = 3. Densitometric analyses obtained from those results are shown as graphs.