Journal: Communications Biology

Article Title: GDE7 produces cyclic phosphatidic acid in the ER lumen functioning as a lysophospholipid mediator

doi: 10.1038/s42003-023-04900-4

Figure Lengend Snippet: a Wild-type (WT), GDE7-deficient (KO) MCF-7 cells, and KO cells overexpressing mGDE7 (RE) were cultured in serum-free medium for 20 h. Total RNA was isolated and analyzed by reverse transcription qPCR for mRNA levels of CD36 , CYP27A1 , and PPARG . Values are expressed as the ratios to the GAPDH levels (mean values ± S.D., n = 4). Dunnett’s test was conducted for analysis. ** P < 0.01, *** P < 0.001 (vs. KO). b 3T3-L1 cells overexpressing hGDE7 (h7) and control 3T3-L1 cells (–) were cultured in serum-free medium with or without 1 μM rosiglitazone (ROSI) for 20 h. Total RNA was isolated and analyzed by reverse transcription qPCR for mRNA levels of Cd36 , Cyp27a1 , Gdpd3 (endogenous GDE7), GDPD3 (exogenous GDE7), Adipoq , Fabp4 , and Pparg . Values are expressed as the ratios to the Gapdh levels (mean values ± S.D., n = 3). Tukey test was conducted for analysis. * P < 0.05, ** P < 0.01, *** P < 0.001. c 3T3-L1 cells overexpressing hGDE7 (h7) and control 3T3-L1 cells (–) were treated with or without 1 μM ROSI in the presence of 10% calf serum for 1 week (medium change every other day). The cell lysates containing equal amounts of proteins were subjected to immunoblotting with anti-PPARγ, -adiponectin, -FABP4, and -CD36 antibodies. GAPDH was used as a loading control. Different blots were used for each antibody. All the experiments were repeated at least twice.

Article Snippet: Anti-PPARγ (#2435), anti-adiponectin (#2789), anti-CD36 (#28109), and anti-FABP4 (#2120) antibodies were from Cell Signaling Technology (Danvers, MA).

Techniques: Cell Culture, Isolation, Reverse Transcription, Control, Western Blot

Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

Article Title: FABP4 regulates eosinophil recruitment and activation in allergic airway inflammation

doi: 10.1152/ajplung.00429.2017

Figure Lengend Snippet: Allergen exposure leads to increased fatty acid binding protein 4 (FABP4) expression in the lungs. A: outline of protocol for cockroach antigen (CRA)-induced allergic airway inflammation in mice. i.p, intraperitoneal; s.c, subcutaneous; i.n, intranasal. B: expression of FABP4 in the lungs of wild-type (WT) mice exposed to CRA challenge (top, middle) or saline alone (control, top) after immunohistochemical staining with anti-FABP4. Airway epithelial cells are shown with black arrows; endothelial cells, black arrowheads; smooth muscle cells, red arrows; adipocytes, red arrowheads; and inflammatory cells, blue arrows. Immunohistochemical staining of lung sections from allergen-challenged WT mice with a control antibody (bottom, middle) and from allergen-challenged FABP4 knockout (KO) mice with FABP4 antibody (bottom) is shown as negative controls. Scale bar = 50 µm. C: dual immunostaining of lung sections from CRA-exposed WT mice with antibodies against FABP4 (green) and eosinophil-specific major basic protein (MBP) (red). Cells positive for FABP4 only are indicated with yellow arrows, and cells positive for FABP4 and MBP are indicated with white arrows. Scale bar = 20 µm. Data representative of 3 mice/group are shown in B and C.

Article Snippet: Western blot analysis was carried out using rabbit polyclonal Ab against FABP4 (0.1 μg/ml, overnight at 4°C) followed by horseradish peroxidase (HRP)-conjugated anti-rabbit IgG (0.16 μg/ml; Jackson ImmunoResearch).

Techniques: Binding Assay, Expressing, Immunohistochemical staining, Staining, Knock-Out, Immunostaining

Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

Article Title: FABP4 regulates eosinophil recruitment and activation in allergic airway inflammation

doi: 10.1152/ajplung.00429.2017

Figure Lengend Snippet: IL-4, TNF-α, and IL-13 induce fatty acid binding protein 4 (FABP4) expression in murine eosinophils. A: effect of inflammatory cytokines on FABP4 expression in mouse eosinophils by qPCR. GM-CSF, granulocyte macrophage colony-stimulating factor. B: effect of IL-4 and IL-13 on FABP4 protein expression in the presence of signal transducer and activator of transcription 6 (STAT6) inhibitor (100 nM) or vehicle (DMSO) examined by Western blot followed by densitometric analysis. C: effect of TNF-α on FABP4 protein expression by Western blot followed by densitometric analysis. Combined data (means ± SE) of 3 experiments are shown. Representative data for Western blot are shown below graph in B and C. *P < 0.03 in A, <0.05 in B and C for control/vehicle/untreated vs. cytokine treated.

Article Snippet: Western blot analysis was carried out using rabbit polyclonal Ab against FABP4 (0.1 μg/ml, overnight at 4°C) followed by horseradish peroxidase (HRP)-conjugated anti-rabbit IgG (0.16 μg/ml; Jackson ImmunoResearch).

Techniques: Binding Assay, Expressing, Western Blot

Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

Article Title: FABP4 regulates eosinophil recruitment and activation in allergic airway inflammation

doi: 10.1152/ajplung.00429.2017

Figure Lengend Snippet: Fatty acid binding protein 4 (FABP4) promotes cell adhesion via regulation of integrin-β2 expression. A and B: adhesion and morphology of wild-type (WT) and FABP4 knockout (KO) eosinophils on recombinant murine (rm) VCAM-1-coated coverslips. C and D: adhesion and morphology of WT and FABP4 KO eosinophils on rm ICAM-1-coated coverslips. Scale bar = 20 µm in B and D. E: rolling of WT and FABP4 KO eosinophils on rm VCAM-1-coated coverslips under conditions of flow (wall shear stress 1.0–2.0 dynes/cm2) in a flow chamber. F: detachment of WT and FABP4 KO eosinophils from rm ICAM-1-coated coverslips under conditions of flow in a flow chamber. G: expression of cell adhesion molecules in WT and FABP4 KO eosinophils by flow cytometry. H: expression of peroxisome proliferator-activated receptor-γ (PPAR-γ) in WT and FABP4 KO eosinophils by qPCR. Combined data (means ± SE) of 3 experiments in duplicate are shown in A, C, E, F, and H. Representative data of 3 independent experiments with eosinophils from 3 different mice/group are shown in B, D, and G. *P < 0.01 in A, C, and F and <0.05 in H for comparison with WT eosinophils.

Article Snippet: Western blot analysis was carried out using rabbit polyclonal Ab against FABP4 (0.1 μg/ml, overnight at 4°C) followed by horseradish peroxidase (HRP)-conjugated anti-rabbit IgG (0.16 μg/ml; Jackson ImmunoResearch).

Techniques: Binding Assay, Expressing, Knock-Out, Recombinant, Flow Cytometry

Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

Article Title: FABP4 regulates eosinophil recruitment and activation in allergic airway inflammation

doi: 10.1152/ajplung.00429.2017

Figure Lengend Snippet: Fatty acid binding protein 4 (FABP4) is required for efficient eosinophil migration. A: in vitro migration of wild-type (WT) and FABP4 knockout (KO) eosinophils toward murine eotaxin-1 (100 nM) across membranes (top) or endothelial cell monolayers (bottom) in Transwell chambers. B: expression of C-C chemokine receptor type 3 (CCR3) in WT and FABP4 KO eosinophils without and with eotaxin-1 treatment (100 nM, 30 min) by flow cytometry. C: FITC-phalloidin staining of WT and FABP4 KO eosinophils adhered on poly-l-lysine and treated with eotaxin-1 (100 nM) for the indicated time points. Scale bar = 20 µm. D: [Ca2+]i levels in WT and FABP4 KO before and after stimulation with eotaxin-1 (top) or ionomycin (bottom) by digital videofluorescence imaging using the Ca2+ indicator dye fura-2 AM. >150 cells were analyzed for each genotype. E: total and phospho-ERK(1/2) expression in WT and FABP4 KO eosinophils treated with eotaxin-1 (100 nM, 15 min) by Western blot analysis followed by densitometric analysis of protein bands. Data represent means ± SD of 2 experiments. F: representative Western blot of total and phospho-ERK(1/2) expression in WT and FABP4 KO eosinophils treated with eotaxin-1 as in E. G: recruitment of intravenously transferred fluorescently labeled WT and FABP4 KO eosinophils to the lungs of A. alternata-challenged WT mice 24 h after infusion evaluated by flow cytometry. Data of individual experiments (n = 5 WT mice, M1–M5, right) and recruitment relative to WT eosinophils (left) are shown. BALF, bronchoalveolar lavage fluid. Combined data (means ± SE) of 3 experiments are shown in A and D, and data representative of 3 independent experiments with eosinophils from 3 different mice/group are shown in B and C. *P < 0.02 in A and G for comparison with WT eosinophils.

Article Snippet: Western blot analysis was carried out using rabbit polyclonal Ab against FABP4 (0.1 μg/ml, overnight at 4°C) followed by horseradish peroxidase (HRP)-conjugated anti-rabbit IgG (0.16 μg/ml; Jackson ImmunoResearch).

Techniques: Binding Assay, Migration, In Vitro, Knock-Out, Expressing, Flow Cytometry, Staining, Imaging, Western Blot, Labeling

Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

Article Title: FABP4 regulates eosinophil recruitment and activation in allergic airway inflammation

doi: 10.1152/ajplung.00429.2017

Figure Lengend Snippet: Fatty acid binding protein 4 (FABP4) deficiency results in attenuation of allergen-induced airway cellular inflammation. A: hematoxylin and eosin (H and E) staining of paraffin-embedded lung sections from saline (control) and cockroach antigen (CRA)-challenged wild-type (WT) and FABP4 knockout (KO) mice. Representative image for each group is shown. Scale bar = 50 µm. B: total cell counts in the bronchoalveolar lavage fluid (BALF) of CRA-challenged and control WT and FABP4 KO mice. C–F: number of eosinophils, macrophages, lymphocytes, and neutrophils, respectively, in the BALF of CRA-challenged and control WT and FABP4 KO mice. Combined data (means ± SE) of mice from 2 to 3 independent experiments are shown in B–F (n = 8–12 mice/group). *P < 0.05 in B–D and <0.02 in E and F for comparison of CRA-challenged groups.

Article Snippet: Western blot analysis was carried out using rabbit polyclonal Ab against FABP4 (0.1 μg/ml, overnight at 4°C) followed by horseradish peroxidase (HRP)-conjugated anti-rabbit IgG (0.16 μg/ml; Jackson ImmunoResearch).

Techniques: Binding Assay, Staining, Knock-Out

Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

Article Title: FABP4 regulates eosinophil recruitment and activation in allergic airway inflammation

doi: 10.1152/ajplung.00429.2017

Figure Lengend Snippet: Mice deficient in fatty acid binding protein 4 (FABP4) exhibit decreased allergen-induced eosinophilia. A and B: infiltrated eosinophils in lung tissue detected by immunohistochemical staining for major basic protein (MBP) (stained dark brown) and quantitation of immunopositive cells. Representative images for control and cockroach antigen (CRA)-challenged wild-type (WT) and FABP4 knockout (KO) mice are shown in A, and the number of positively stained cells in randomly selected nonoverlapping microscopic fields (18 ± 4) counted at ×400 magnification is shown in B. Scale bar = 50 µm. C and D: percentage of eosinophils in peripheral blood and bone marrow (BM), respectively, of control and CRA-challenged WT and FABP4 KO mice determined based on cell morphology after Hema3 staining. Combined data (means ± SE) of mice from 2 to 3 independent experiments are shown (n = 8–12 mice/group in B and D and 5 mice/group in C). *P < 0.01 in B and <0.05 in C and D for comparison of CRA-challenged groups.

Article Snippet: Western blot analysis was carried out using rabbit polyclonal Ab against FABP4 (0.1 μg/ml, overnight at 4°C) followed by horseradish peroxidase (HRP)-conjugated anti-rabbit IgG (0.16 μg/ml; Jackson ImmunoResearch).

Techniques: Binding Assay, Immunohistochemical staining, Staining, Quantitation Assay, Knock-Out

Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

Article Title: FABP4 regulates eosinophil recruitment and activation in allergic airway inflammation

doi: 10.1152/ajplung.00429.2017

Figure Lengend Snippet: Decreased levels of proinflammatory mediators in the lungs of allergen-challenged fatty acid binding protein 4 (FABP4)-deficient mice. A and B: Th1-Th2 cytokine and TNF-α levels in bronchoalveolar lavage fluid (BALF) and lung lysates of control and cockroach antigen (CRA)-challenged wild-type (WT) and FABP4 knockout (KO) mice, respectively. C: eotaxin-1 and -2 levels in BALF of above groups of mice. D: cysteinyl leukotriene C4 (LTC4) levels in lung tissue of above groups of mice. Combined data (means ± SE) of n = 9–10 mice/group in A, 4–8 mice/group in B, 7–9 mice/group in C, and 3–4 mice/group in D are shown. *P < 0.03 in A and B and <0.05 in D for comparison of CRA-challenged groups.

Article Snippet: Western blot analysis was carried out using rabbit polyclonal Ab against FABP4 (0.1 μg/ml, overnight at 4°C) followed by horseradish peroxidase (HRP)-conjugated anti-rabbit IgG (0.16 μg/ml; Jackson ImmunoResearch).

Techniques: Binding Assay, Knock-Out

Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

Article Title: FABP4 regulates eosinophil recruitment and activation in allergic airway inflammation

doi: 10.1152/ajplung.00429.2017

Figure Lengend Snippet: Airway hyperresponsiveness (AHR), mucus secretion, and smooth muscle thickening are attenuated in allergen-challenged fatty acid binding protein 4 (FABP4)-deficient mice. A: invasive measurement of airway responsiveness following exposure to increasing concentrations of aerosolized methacholine (MCh) in mechanically ventilated control and cockroach antigen (CRA)-challenged wild-type (WT) and FABP4 knockout (KO) mice. B and C: airway mucus secretion in the above groups of mice assessed by periodic acid-Schiff (PAS) staining (stained dark-pink, black arrows) and quantitation of PAS-positive area (4–5 airways/mouse with perimeter 730 ± 20 μm). Representative images are shown in B. Scale bar = 50 µm. D and E: airway smooth muscle thickening in control and allergen-challenged mice assessed by immunohistochemical staining for α-smooth muscle actin (α-SMA) expression (stained brown, black arrows) and quantitation of α-SMA-positive area (3–4 airways/mouse with perimeter 710 ± 20 μm). Representative images are shown in D. Scale bar = 50 µm. Combined data (means ± SE) of n = 6–8 mice/group for AHR, 8–10 mice/group for PAS, and 7–9 mice/group for α-SMA are shown. *P < 0.04 in A, and <0.025 in C and E for comparison of CRA-challenged groups.

Article Snippet: Western blot analysis was carried out using rabbit polyclonal Ab against FABP4 (0.1 μg/ml, overnight at 4°C) followed by horseradish peroxidase (HRP)-conjugated anti-rabbit IgG (0.16 μg/ml; Jackson ImmunoResearch).

Techniques: Binding Assay, Knock-Out, Staining, Quantitation Assay, Immunohistochemical staining, Expressing

Journal: International Journal of Molecular Sciences

Article Title: Adipocytes in the Uterine Wall during Experimental Healing and in Cesarean Scars during Pregnancy

doi: 10.3390/ijms242015255

Figure Lengend Snippet: The healing area of the operated rat uterine horn. ( A ) ×50, Mallory staining; ( B ) ×100, immunohistochemical staining on FABP4; ( C ) ×100, immunohistochemical staining on α-SMA, Mayer’s hematoxylin staining; ( D ) ×100, CD34 immunohistochemical staining, Mayer’s hematoxylin staining; ( E ) ×100, CD68 immunohistochemical staining, Mayer’s hematoxylin staining; ( F ) ×200, CD163 immunohistochemical staining, Mayer’s hematoxylin staining; ( G ) ×200, CD206 immunohistochemical staining, Mayer’s hematoxylin staining; ( A1 , B1 , C1 , D1 , E1 , F1 , G1 ) 7th day after surgery; ( A2 , B2 , C2 , D2 , E2 , F2 , G2 ) 30th day; ( A3 , A4 ) 60th day; ( H1 ) The crown-like structures in the area of attachment of adipose tissue to the uterine wall; ( H2 ) CD68 cells in the adipose tissue attached to the uterine wall on the 7th, 30th, and 60th days after surgery; ( H3 ) CD206 cells in the adipose tissue attached to the uterine wall on the 7th, 30th, and 60th day after surgery; ( H4 ) CD163 cells in the adipose tissue attached to the uterine wall on the 7th, 30th, and 60th days after surgery; ( H5 ) CD34 cells in the adipose tissue attached to the uterine wall on the 7th, 30th and 60th days after surgery; (*) p < 0.05; (**) p < 0.01; ( ● ) The blue dot presents value for individual sample on the 7th day after the operation; ( ● ) The red dot presents value for individual sample on the 30th day after the operation; ( ● ) The green dot presents value for individual sample on the 60th day after the operation. The blue, red, green horizontal lines present the medians.

Article Snippet: For clinical specimens, we used mouse antibody (mAb) to cytokeratin 8 (cat# DB098-RTU, DB Biotech, Kosice, Slovakia, EU) for the detection of epithelial cells, mouse mAb to α-SMA (#ab7817, Abcam) for smooth muscle cells, rabbit mAb to CD34 (#ab81289, Abcam) for endothelial cells, rabbit mAb t to FABP4 (#ab219595, Abcam) for adipocytes, mouse mAb to CD68 (#ab783, Abcam), rabbit mAb to CD163 (#ab182422, Abcam), and rabbit polyclonal antibodies to CD206 (#ab64693, Abcam) for macrophages.

Techniques: Staining, Immunohistochemistry

Journal: International Journal of Molecular Sciences

Article Title: Adipocytes in the Uterine Wall during Experimental Healing and in Cesarean Scars during Pregnancy

doi: 10.3390/ijms242015255

Figure Lengend Snippet: The uterine wall of women near the placenta. ( A , B ) Mallory staining; ( A1 , B1 , C1 , D1 ) Uterus without previous cesarean section and uterine scar; ( A2 , B2 , C2 , D2 ) Uterus with caesarian scar and non-complicated pregnancy; ( A3 , B3 , C3 , D3 ) Uterus with PAS; ( A1 , A3 , B3,D1 – D3 ) ×50; ( A2 , B2, C2 , C3 ) ×100; ( C1 ) ×200; ( C1 – C3 ) CD68 immunohistochemical staining, Mayer’s hematoxylin staining; ( D1 – D3 ) FABP4 immunohistochemical staining, Mayer’s hematoxylin staining; ( E1 ) CD68 cells in comparison groups; ( E2 ) Adipocyte clusters in comparison groups; norma—the control group of women without previous cesarean section and uterine scar; scar—the group of women with a cesarean scar and non-complicated pregnancy; PAS—a group of women with PAS; (**) p < 0.01; (****) p < 0.001; (ns) not significant; ( ● ) The blue dot presents value for individual sample of the control group of women without previous cesarean section and uterine scar; ( ● ) The red dot presents value for individual sample of the group of women with a cesarean scar and non-complicated pregnancy; ( ● ) The green dot presents value for individual sample of a group of women with PAS. The blue, red, green horizontal lines present the medians.

Article Snippet: For clinical specimens, we used mouse antibody (mAb) to cytokeratin 8 (cat# DB098-RTU, DB Biotech, Kosice, Slovakia, EU) for the detection of epithelial cells, mouse mAb to α-SMA (#ab7817, Abcam) for smooth muscle cells, rabbit mAb to CD34 (#ab81289, Abcam) for endothelial cells, rabbit mAb t to FABP4 (#ab219595, Abcam) for adipocytes, mouse mAb to CD68 (#ab783, Abcam), rabbit mAb to CD163 (#ab182422, Abcam), and rabbit polyclonal antibodies to CD206 (#ab64693, Abcam) for macrophages.

Techniques: Staining, Immunohistochemistry, Comparison

Journal: International Journal of Molecular Sciences

Article Title: Adipocytes in the Uterine Wall during Experimental Healing and in Cesarean Scars during Pregnancy

doi: 10.3390/ijms242015255

Figure Lengend Snippet: The uteroplacental region with PAS, ×50. FABP4 immunohistochemical staining, Mayer’s hematoxylin staining; ( A ) Villi invaded the myometrium in the scar area, а cluster of adipocytes; ( B ) Adipocyte clusters in the perivascular space; ( C ) Adipocyte clusters in the perivascular space (continuation of the uterine wall shown in ( B )); black arrow indicates a single adipocyte.

Article Snippet: For clinical specimens, we used mouse antibody (mAb) to cytokeratin 8 (cat# DB098-RTU, DB Biotech, Kosice, Slovakia, EU) for the detection of epithelial cells, mouse mAb to α-SMA (#ab7817, Abcam) for smooth muscle cells, rabbit mAb to CD34 (#ab81289, Abcam) for endothelial cells, rabbit mAb t to FABP4 (#ab219595, Abcam) for adipocytes, mouse mAb to CD68 (#ab783, Abcam), rabbit mAb to CD163 (#ab182422, Abcam), and rabbit polyclonal antibodies to CD206 (#ab64693, Abcam) for macrophages.

Techniques: Immunohistochemistry, Staining

Journal: International Journal of Molecular Sciences

Article Title: Adipocytes in the Uterine Wall during Experimental Healing and in Cesarean Scars during Pregnancy

doi: 10.3390/ijms242015255

Figure Lengend Snippet: Vascular collectors of the uteroplacental region in the area of placental villus invasion. ( A ). Mallory staining, ×50; ( B , C ). CD34 immunohistochemical staining, ×100 ( B ), ×200 ( C ); ( D , E ). FABP4 immunohistochemical staining, ×200 ( D ), ×400 ( E ).

Article Snippet: For clinical specimens, we used mouse antibody (mAb) to cytokeratin 8 (cat# DB098-RTU, DB Biotech, Kosice, Slovakia, EU) for the detection of epithelial cells, mouse mAb to α-SMA (#ab7817, Abcam) for smooth muscle cells, rabbit mAb to CD34 (#ab81289, Abcam) for endothelial cells, rabbit mAb t to FABP4 (#ab219595, Abcam) for adipocytes, mouse mAb to CD68 (#ab783, Abcam), rabbit mAb to CD163 (#ab182422, Abcam), and rabbit polyclonal antibodies to CD206 (#ab64693, Abcam) for macrophages.

Techniques: Staining, Immunohistochemistry