Journal: Journal of Immunology Research

Article Title: Protective Effects of Thalidomide on High-Glucose-Induced Podocyte Injury through In Vitro Modulation of Macrophage M1/M2 Differentiation

doi: 10.1155/2020/8263598

Figure Lengend Snippet: Effects of thalidomide on iNOS, CD206, Arg-1 and TNF- α protein expression in 33.3 mM glucose-induced macrophage. (a) iNOS and CD206 protein expressions. (d) Arg-1 protein expression. (f) TNF- α protein expression. The results of iNOS, CD206, Arg-1, and TNF- α were represented in (b), (c), (e), and (g), respectively. All results were expressed as a ration with respect to control and represented as the mean ± SD in triplicates. ∗ p < 0.05; versus 11.1 mM glucose. # p < 0.05; versus 33.3 mM glucose. & p < 0.05; versus Tha100. ^ p < 0.05; versus Tha50. Abbreviations: LPS: lipopolysaccharide; Tha50: 50 μ g/ml thalidomide in 33.3 mM glucose; Tha100: 100 μ g/ml thalidomide in 33.3 mM glucose; Tha200: 200 μ g/ml thalidomide in 33.3 mM glucose; iNOS: inducible nitric oxide synthase; CD206: mannose receptor; TNF- α : tumor necrosis factor- α ; Arg-1: arginase-1.

Article Snippet: After several consecutive rinses with a washing buffer (0.1% Tween-20 in PBS), the membranes were incubated with primary antibodies against iNOS at 1 : 500 dilution (Catalog No. BA0362, Boster), TNF- α (Catalog No. BA0131, Boster) at 1 : 500 dilution, CD206 (Catalog No. A02285-2, Boster) at 1 : 500 dilution, and antibody against Arg-1 (Catalog No. BM4000, Boster) at 1 : 500 dilution overnight at 4°C.

Techniques: Expressing, Control

Journal: Journal of Immunology Research

Article Title: Protective Effects of Thalidomide on High-Glucose-Induced Podocyte Injury through In Vitro Modulation of Macrophage M1/M2 Differentiation

doi: 10.1155/2020/8263598

Figure Lengend Snippet: Effects of thalidomide on iNOS, CD206, Arg-1, and TNF- α mRNA expressions in 33.3 mM glucose-induced macrophage. (a) iNOS mRNA expression. (b) CD206 mRNA expression. (c) CD206 mRNA expression. (d) TNF- α mRNA expression.All the results were represented as the mean ± SD in triplicates ∗ p < 0.05; versus 11.1 mM glucose. # p < 0.05; versus 33.3 mM glucose. & p < 0.05; versus Tha100. ^ p < 0.05; versus Tha50. Abbreviations: LPS: lipopolysaccharide; Tha50: 50 μ g/ml thalidomide in 33.3 mM glucose; Tha100: 100 μ g/ml thalidomide in 33.3 mM glucose; Tha200: 200 μ g/ml thalidomide in 33.3 mM glucose; iNOS: inducible nitric oxide synthase; CD206: mannose receptor; TNF- α : tumor necrosis factor- α ; Arg-1: arginase-1.

Article Snippet: After several consecutive rinses with a washing buffer (0.1% Tween-20 in PBS), the membranes were incubated with primary antibodies against iNOS at 1 : 500 dilution (Catalog No. BA0362, Boster), TNF- α (Catalog No. BA0131, Boster) at 1 : 500 dilution, CD206 (Catalog No. A02285-2, Boster) at 1 : 500 dilution, and antibody against Arg-1 (Catalog No. BM4000, Boster) at 1 : 500 dilution overnight at 4°C.

Techniques: Expressing

Journal: American Journal of Translational Research

Article Title: The distribution of macrophage subtypes and their relationship to bone morphogenetic protein 2 in calcified aortic valve stenosis

doi:

Figure Lengend Snippet: Representative immunostaining images showing the expression of macrophage subtype markers [CD68 (A), CD163 (B), CD206 (C), heme oxygenase-1 (D), and inducible nitric oxide synthase (iNOS) (E)] and bone morphogenetic protein 2 (BMP2) (F) by single immunohistochemistry in the valve center part and spongiosa layer of a single case in the calcification group. Cells of the iNOS+ M1 and CD163+/CD206+ M2 subtypes frequently increased, while cells of the CD68+ M1/M2 subtype and heme oxygenase-1+ Mox subtypes were relatively few. Note that scattered BMP2+ cells were also present. iNOS and BMP2 were present in some vascular endothelial cells.

Article Snippet: For IHC, formalin-fixed and paraffin-embedded tissue sections (4 μm in thickness) were blocked with 0.3% H 2 O 2 in methanol at 4°C for 30 min and then subjected to antigen retrieval in trypsin for 30 min at 37°C for anti-CD68 antibody, in citric acid (Antigen Retrieval Solution pH 6; Iatron Laboratories Inc., Tokyo, Japan) for anti-CD 163 antibody, anti-iNOS antibody, anti-HO-1 antibody, and anti-OPN antibody, and in EDTA (pH 9.0; Nichirei, Tokyo, Japan) in an autoclave (2 atmospheres, 121°C, 20 min) for anti-CD206 antibody.

Techniques: Immunostaining, Expressing, Immunohistochemistry

Journal: American Journal of Translational Research

Article Title: The distribution of macrophage subtypes and their relationship to bone morphogenetic protein 2 in calcified aortic valve stenosis

doi:

Figure Lengend Snippet: Frequency of cells immunopositive for macrophage subtype markers and bone morphogenetic factor-2 (BMP2) in the calcification and noncalcification groups

Article Snippet: For IHC, formalin-fixed and paraffin-embedded tissue sections (4 μm in thickness) were blocked with 0.3% H 2 O 2 in methanol at 4°C for 30 min and then subjected to antigen retrieval in trypsin for 30 min at 37°C for anti-CD68 antibody, in citric acid (Antigen Retrieval Solution pH 6; Iatron Laboratories Inc., Tokyo, Japan) for anti-CD 163 antibody, anti-iNOS antibody, anti-HO-1 antibody, and anti-OPN antibody, and in EDTA (pH 9.0; Nichirei, Tokyo, Japan) in an autoclave (2 atmospheres, 121°C, 20 min) for anti-CD206 antibody.

Techniques:

Journal: American Journal of Translational Research

Article Title: The distribution of macrophage subtypes and their relationship to bone morphogenetic protein 2 in calcified aortic valve stenosis

doi:

Figure Lengend Snippet: Evaluation of the expression of bone morphogenetic protein 2 (BMP2) in macrophage subtypes by double immunofluorescence staining. Because the anti-iNOS antibody and the anti-BMP2 antibody were both rabbit polyclonal antibodies, single immunofluorescence staining was performed for each of the two proteins using serial sections. Nuclei were counterstained with 4’,6-diamidino-2-phenylindole (blue). Co-expression of BMP2 (green) was confirmed in a subset of the cells that showed positive staining for CD68 (M1/M2 subtype), CD206 (M2 subtype), and heme oxygenase-1 (Mox subtype) (red). However, co-expression of iNOS (M1 subtype) and BMP2 was not observed.

Article Snippet: For IHC, formalin-fixed and paraffin-embedded tissue sections (4 μm in thickness) were blocked with 0.3% H 2 O 2 in methanol at 4°C for 30 min and then subjected to antigen retrieval in trypsin for 30 min at 37°C for anti-CD68 antibody, in citric acid (Antigen Retrieval Solution pH 6; Iatron Laboratories Inc., Tokyo, Japan) for anti-CD 163 antibody, anti-iNOS antibody, anti-HO-1 antibody, and anti-OPN antibody, and in EDTA (pH 9.0; Nichirei, Tokyo, Japan) in an autoclave (2 atmospheres, 121°C, 20 min) for anti-CD206 antibody.

Techniques: Expressing, Double Immunofluorescence Staining, Immunofluorescence, Staining

Journal: American Journal of Translational Research

Article Title: The distribution of macrophage subtypes and their relationship to bone morphogenetic protein 2 in calcified aortic valve stenosis

doi:

Figure Lengend Snippet: Evaluation of the expression of CD206 by cells of the M2 subtype, heme oxygenase-1 (HO-1) by cells of the Mox subtype, and bone morphogenetic protein 2 (BMP2) mRNAs in the aortic valve by reverse transcription polymerase chain reaction. CD206 mRNA was expressed in 8 cases (80%) in the calcification group and in 3 cases (60%) in the noncalcification group. HO-1 and BMP2 mRNAs were expressed in 6 cases (60%) and 5 cases (50%), respectively, in the calcification group and in 4 cases (80%) and 4 cases (80%), respectively, in the noncalcification group. In cells that expressed BMP2 mRNA, expression of mRNA of CD206 and/or HO-1 was observed. Lanes 1-10, calcification group; N1-N5, noncalcification group. M, marker; PC, positive control; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

Article Snippet: For IHC, formalin-fixed and paraffin-embedded tissue sections (4 μm in thickness) were blocked with 0.3% H 2 O 2 in methanol at 4°C for 30 min and then subjected to antigen retrieval in trypsin for 30 min at 37°C for anti-CD68 antibody, in citric acid (Antigen Retrieval Solution pH 6; Iatron Laboratories Inc., Tokyo, Japan) for anti-CD 163 antibody, anti-iNOS antibody, anti-HO-1 antibody, and anti-OPN antibody, and in EDTA (pH 9.0; Nichirei, Tokyo, Japan) in an autoclave (2 atmospheres, 121°C, 20 min) for anti-CD206 antibody.

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Marker, Positive Control

Journal: American Journal of Translational Research

Article Title: The distribution of macrophage subtypes and their relationship to bone morphogenetic protein 2 in calcified aortic valve stenosis

doi:

Figure Lengend Snippet: Scheme of a possible mechanism of aortic valve calcification. Noncalcified valves contain a few macrophages, predominantly those of the M1 subtype [inducible nitric oxide synthase (iNOS)+/CD68+] and, less frequently, cells of the M2 subtype (CD68+/CD163+/CD206+). In the pre-calcification phase, against a background of fibrosis, M1 and M2 subtypes gradually accumulate, with a mildly increasing ratio of cells of the M2 subtype to those of the M1 subtype. A few heme oxygenase (HO)-1+ Mox appear. In the calcified valve, calcification is accompanied by the presence of osteoblast-like cells (OLC). In this valve, all subtypes of macrophages are increasing, with a relatively high ratio of cells of the HO-1+ Mox subtype. Some cells of the M2 and Mox subtypes express bone morphogenic protein-2 (BMP2).

Article Snippet: For IHC, formalin-fixed and paraffin-embedded tissue sections (4 μm in thickness) were blocked with 0.3% H 2 O 2 in methanol at 4°C for 30 min and then subjected to antigen retrieval in trypsin for 30 min at 37°C for anti-CD68 antibody, in citric acid (Antigen Retrieval Solution pH 6; Iatron Laboratories Inc., Tokyo, Japan) for anti-CD 163 antibody, anti-iNOS antibody, anti-HO-1 antibody, and anti-OPN antibody, and in EDTA (pH 9.0; Nichirei, Tokyo, Japan) in an autoclave (2 atmospheres, 121°C, 20 min) for anti-CD206 antibody.

Techniques:

Journal: Translational Oncology

Article Title: Methionine restriction promotes the polarization of macrophages towards M1 and the immunotherapy effect of PD-L1/PD-1 blockades by inhibiting the secretion of MIF by gastric carcinoma cells

doi: 10.1016/j.tranon.2024.102181

Figure Lengend Snippet: The primer sequences.

Article Snippet: Slices were then treated with Triton-100 and 5 % BSA, followed by incubation with the anti-CD86 antibody (#SAB5701086, 1:80, Merck), anti-CD206 antibody (#ZRB1440, Merck), or anti-CD8a Antibody (#MABF1983M, Merck).

Techniques: Sequencing

Journal: Translational Oncology

Article Title: Methionine restriction promotes the polarization of macrophages towards M1 and the immunotherapy effect of PD-L1/PD-1 blockades by inhibiting the secretion of MIF by gastric carcinoma cells

doi: 10.1016/j.tranon.2024.102181

Figure Lengend Snippet: Effects of methionine restriction on M1 polarization and M2 polarization of macrophages. Gastric carcinoma cells (MKN45 and AGS) transfected with LV-NC or LV-METase were co-cultured with M0 macrophages. N = 3. (A-B) The expression of M1 macrophage markers (CD86, TNF-ɑ, IL-12B) was measured by qRT-PCR. (C-D) The expression of M2 macrophage markers (CD206, CD204, Arg-1) was measured by qRT-PCR. The content of M1 macrophages (E-H) and M2 macrophages (I-L) was detected by flow cytometry. ⁎⁎ P < 0.01, ⁎⁎⁎ P < 0.001 vs. M0 + GC-LV-NC. A student's t -test was performed.

Article Snippet: Slices were then treated with Triton-100 and 5 % BSA, followed by incubation with the anti-CD86 antibody (#SAB5701086, 1:80, Merck), anti-CD206 antibody (#ZRB1440, Merck), or anti-CD8a Antibody (#MABF1983M, Merck).

Techniques: Transfection, Cell Culture, Expressing, Quantitative RT-PCR, Flow Cytometry

Journal: Translational Oncology

Article Title: Methionine restriction promotes the polarization of macrophages towards M1 and the immunotherapy effect of PD-L1/PD-1 blockades by inhibiting the secretion of MIF by gastric carcinoma cells

doi: 10.1016/j.tranon.2024.102181

Figure Lengend Snippet: Effect of methionine restriction on MIF secretion in gastric carcinoma cells. Gastric carcinoma cells (MKN45, MFC, and AGS) were transfected with LV-NC or LV-METase. N = 3. (A) The mRNA levels of MIF were detected by qRT-PCR. (B) The protein levels of MIF and METase (HA-METase) were detected by western blot. (C) The secretion of MIF in the supernatant of gastric carcinoma cells was detected by ELISA. Gastric carcinoma cells (MKN45 and AGS) transfected with LV-NC or LV-METase were added with anti-MIF or IgG, and co-cultured with M0 macrophages. (D-F) The expression of M1 macrophage markers (CD86, TNF-ɑ, IL-12B) was measured by qRT-PCR. (G-I) The expression of M2 macrophage markers (CD206, CD204, Arg-1) was measured by qRT-PCR. The content of M1 macrophages (J) and M2 macrophages (K) was detected by flow cytometry. ns P >0.05, * P < 0.05, ⁎⁎ P < 0.01, ⁎⁎⁎ P < 0.001. A two-way analysis of variance followed by Sidak's multiple comparisons test was performed.

Article Snippet: Slices were then treated with Triton-100 and 5 % BSA, followed by incubation with the anti-CD86 antibody (#SAB5701086, 1:80, Merck), anti-CD206 antibody (#ZRB1440, Merck), or anti-CD8a Antibody (#MABF1983M, Merck).

Techniques: Transfection, Quantitative RT-PCR, Western Blot, Enzyme-linked Immunosorbent Assay, Cell Culture, Expressing, Flow Cytometry

Journal: Translational Oncology

Article Title: Methionine restriction promotes the polarization of macrophages towards M1 and the immunotherapy effect of PD-L1/PD-1 blockades by inhibiting the secretion of MIF by gastric carcinoma cells

doi: 10.1016/j.tranon.2024.102181

Figure Lengend Snippet: . Effect of methionine restriction on tumor formation and M2/M1 polarization in vivo. MFC cells transfected with LV-NC or LV-METase were injected into mice. N = 5. (A-C) The volume and weight of the tumor. (D) The protein levels of METase (HA-METase) were detected by western blot. (E-F) The content of M1 and M2 macrophages was detected by flow cytometry. (G) The immunohistochemical staining of CD86 (M1 macrophage marker) and CD206 (M2 macrophage marker) was performed. Scale bar: 40 μm. * P < 0.05, ⁎⁎⁎ P < 0.001 vs LV-NC. (A) A two-way analysis of variance followed by Sidak's multiple comparisons test was performed. (C/E/F) A student's t -test was performed.

Article Snippet: Slices were then treated with Triton-100 and 5 % BSA, followed by incubation with the anti-CD86 antibody (#SAB5701086, 1:80, Merck), anti-CD206 antibody (#ZRB1440, Merck), or anti-CD8a Antibody (#MABF1983M, Merck).

Techniques: In Vivo, Transfection, Injection, Western Blot, Flow Cytometry, Immunohistochemical staining, Staining, Marker