Journal: Clinical and Experimental Pharmacology & Physiology

Article Title: Mechanisms of IL‐17A Neutralisation in Alleviating Renal Fibrosis and Inflammation in Spontaneously Hypertensive Rats

doi: 10.1111/1440-1681.70116

Figure Lengend Snippet: Effects of IL‐17A neutralisation on macrophage polarisation in SHR renal tissues. (A) Representative IHC staining of M1 macrophage markers (iNOS and CD86) with quantitative analysis of their positive areas. (B) Representative IHC staining of M2 macrophage markers (Arg‐1 and CD163) with quantitative analysis of their positive areas. (C) Representative immunoblots and relative expression levels of iNOS, CD86, Arg‐1, and CD163 proteins. (D) Proportion of CD86 + and CD163 + cells among CD68 + macrophages. (E) mRNA expression levels of iNOS, CD86, Arg‐1, and CD163. Data are presented as mean ± SD ( n = 6).

Article Snippet: Sections were then incubated overnight at 4°C with primary antibodies against: E‐cadherin (Boster, China), Collagen III (Boster, China), inducible nitric oxide synthase (iNOS) (Boster, China), CD86 (Boster, China), arginase‐1 (Arg‐1) (Boster, China), CD163 (Boster, China), α‐smooth muscle actin (α‐SMA) (Cell Signalling Technology, USA).

Techniques: Immunohistochemistry, Western Blot, Expressing

Journal: Molecular cell

Article Title: Deciphering functional tumor-immune crosstalk through highly multiplexed imaging and deep visual proteomics.

doi: 10.1016/j.molcel.2024.12.023

Figure Lengend Snippet: Figure 3. T cell subsets have distinctive spatial proteome signatures in colorectal tumor microenvironment (A and B) Ranking of protein quantified in cytotoxic T cells (CTLs) (A) and helper T cells (TH) (B), ranked by median transformed intensity values detected by mass spectrometry, with cell-type-specific markers highlighted. (C and D) Volcano plots illustrating proteomic comparisons of CTLs (C) and TH (D) between the lamina propria and tumor epithelial regions, with T cell-activity- related markers highlighted. Significantly enriched proteins are denoted as yellow or green dots (two-sided t test, false discovery rate [FDR] < 0.05, S0 = 0.1, n = 3). (E and F) Gene Ontology (GO) term analysis of significantly altered proteins in CTLs (E) and TH (F) residing in the lamina propria and tumor epithelial regions.

Article Snippet: For CD4 + ARG1 staining, the sections were incubated overnight at 4 C with primary antibodies (anti-CD4-FITC, 1:100, R&D FAB8165G; anti-ARG1-PE, 1:200,Miltenyi Biotec 130-127-839) in a humidified chamber.

Techniques: Transformation Assay, Mass Spectrometry, Activity Assay

Journal: Molecular cell

Article Title: Deciphering functional tumor-immune crosstalk through highly multiplexed imaging and deep visual proteomics.

doi: 10.1016/j.molcel.2024.12.023

Figure Lengend Snippet: Figure 5. Cytotoxic T cells (CTLs) exhibit close proximity to tumors with a suppressive impact on tumor proliferation (A) Density gradient in the background indicates distance to tumor islands, with brighter yellow representing greater distance from tumor cells. Left panel only has the gradient whereas the middle, zoomed panel additionally depicts yellow masks of the CTLs and the right panel red dots of helper T cells (TH), respectively. (B) Representative images showcasing markers for T cell phenotyping. (C) T cell gating based on the expression of CD3, CD8, CD45RA, CD45RO, and PD1 to analyze the corresponding T cell subpopulations. (D) Representative images showing CD3- and CD8-positive CTLs in tumor parenchyma (TIL-CTLs) with absent CD45RA and CD45RO expression. (E) Distance heatmap depicting proximity and interactions between tumor cells and T and B lymphocyte subtypes, with lower values indicating higher proximity as in Figure 4E. (F) Histogram of Ki67/Ki67+ tumor cell number as a function of distance from RARO CTLs. Data represent three technical replicates. Statistical significance was determined using one-way ANOVA. ***p < 0.001, **p < 0.01. (G) Representative image displaying masks of tumor cells at varying proximities to RARO TIL-CTLs.

Article Snippet: For CD4 + ARG1 staining, the sections were incubated overnight at 4 C with primary antibodies (anti-CD4-FITC, 1:100, R&D FAB8165G; anti-ARG1-PE, 1:200,Miltenyi Biotec 130-127-839) in a humidified chamber.

Techniques: Expressing

Journal: Neural Regeneration Research

Article Title: Fasudil-modified macrophages reduce inflammation and regulate the immune response in experimental autoimmune encephalomyelitis

doi: 10.4103/1673-5374.379050

Figure Lengend Snippet: Fasudil-modified macrophages affect macrophage polarization. A mouse model of chronic EAE was induced by subcutaneous injection of myelin oligodendrocyte glycoprotein peptde 35–55. Macrophages from EAE mice isolated with magnetic beads were treated with 15 μg/mL of Fasudil at 37°C in 5% CO 2 atmosphere for 72 hours. Fasudil-treated macrophages were suspended in PBS at a concentration of 2 × 10 7 cells/200 μL and intraperitoneally injected into EAE mice on days 3 and 11 p.i.. PBS-treated macrophages were used as a control. The mice were randomly divided into two groups: the Fasudil-macrophage group (Fasudil-Mφ) ( n = 12) and the PBS-macrophage group (PBS-Mφ) ( n = 12). On day 28 p.i., splenic MNCs were prepared and stained with antibodies to F4/80 (for macrophages), CD16/32, iNOS, and IL-12 (for pro-inflammatory M1 macrophages), and CD206, Arg-1, and IL-10 (for anti-inflammatory M2 macrophages), and the macrophage subsets were analyzed by flow cytometry. Injection with Fasudil-modified macrophages reduced the percentages of CD16/32 + , iNOS + , and IL-12 + pro-inflammatory M1 macrophages and increased the percentages of Arg-1 + , IL-10 + , and CD206 + anti-inflammatory M2 macrophages. Quantitative results are shown for five mice from each group. The results are expressed as the percentage of double positive cells in the four-quadrant diagram. Data are presented as the mean ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.001 (Student’s t -test). Arg-1: Arginase-1; EAE: experimental autoimmune encephalomyelits; IL-10: interleukin-10; IL-12: interleukin-12; iNOS: inducible nitric oxide synthase; MNCs: mononuclear cells; Mφ: macrophage; PBS: phosphate buffer saline; p.i.: postimmunization.

Article Snippet: The antibodies used for western blotting were as follows: mouse anti-ROCK-II (1:1000; BD Biosciences, Cat# 610624, RRID: AB_397956), rabbit anti-myosin light chain phosphatase target subunit 1 (p-MYPT1)-Thr696 (1:1000; Millipore, Tullagreen Carrigtwohill, Cork, Ireland, Cat# ABS45, RRID: AB_10562238), rabbit anti-iNOS (1:1000, Enzo Life Sciences, Cat# ADI-KAS-NO001, RRID: AB_10622409), mouse anti-Arg-1 (1:1000, BD Biosciences, Cat# 610708, RRID: AB_398031), rabbit anti-toll like receptor 2 (TLR-2) (1:1000; Cell Signaling Technology, Cat# 2229, RRID: AB_2204442), rabbit anti-TLR-4 (1:1000, Cell Signaling Technology, Cat# 14358, RRID: AB_2798460), rabbit anti-myeloid differentiation factor 88 (Myd88) (1:1000; Abcam, Cambridge, UK; Cat# ab2064, RRID: AB_302807), rabbit anti-p-Jun N-terminal kinase (p-JNK) (1:1000, Cell Signaling Technology, Cat# 4668, RRID: AB_823588), mouse anti-p-extracellular regulated protein kinases (p-ERK) (1:1000; Santa Cruz Biotechnology, Dallas, TX, USA, Cat# sc-81492, RRID: AB_1125801), mouse anti-p-P38 (1:1000; Santa Cruz Biotechnology, Cat# sc-7973, RRID: AB_670359), rabbit anti-p-nuclear factor kappa B (p-NF-κB) (1:1000, Cell Signaling Technology, Cat# 3037, RRID: AB_2341216), rabbit anti-ribosomal protein S3 (RPS3) (1:1000, Abcam, Cat# ab128995, RRID: AB_11145466), anti-GAPDH (1:10,000, Cell Signaling Technology, Cat# 2118, RRID: AB_561053), anti-mouse IgG HRP-linked secondary antibody (1:10,000, Cell Signaling Technology, Cat# 7076, RRID: AB_330924), and anti-rabbit IgG HRP-linked secondary antibody (1:10,000, Cell Signaling Technology, Cat# 7074, RRID: AB_2099233).

Techniques: Modification, Injection, Isolation, Magnetic Beads, Concentration Assay, Staining, Flow Cytometry, Saline

Journal: Neural Regeneration Research

Article Title: Fasudil-modified macrophages reduce inflammation and regulate the immune response in experimental autoimmune encephalomyelitis

doi: 10.4103/1673-5374.379050

Figure Lengend Snippet: Fasudil affects macrophage polarization in vitro . A mouse model of chronic EAE was induced by subcutaneous injection of myelin oligodendrocyte glycoprotein peptde 35–55. Macrophages from EAE mice isolated with magnetic beads were treated with 15 μg/mL of Fasudil at 37°C in 5% CO 2 atmosphere for 72 hours. Cells were labeled with antibodies to CD16/32, iNOS, IL-12, CD40, CD206, IL-10, Arg-1, and CD14 and analyzed by flow cytometry. Decreased levels of the pro-inflammatory M1 macrophage subtypes CD16/32, iNOS, and IL-12 and increased levels of the anti-inflammatory M2 macrophage subtypes CD206, IL-10, Arg-1, and CD14 were observed in response to Fasudil treatment. Quantitative data are shown for five exemplars from each group and are reported as mean fluorescence intensity (MFI). Data are presented as the mean ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.001 (Student’s t -test). Arg-1: Arginase-1; EAE: experimental autoimmune encephalomyelits; IL-10:interleukin-10; IL-12:interleukin-12; iNOS: inducible nitric oxide synthase.

Article Snippet: The antibodies used for western blotting were as follows: mouse anti-ROCK-II (1:1000; BD Biosciences, Cat# 610624, RRID: AB_397956), rabbit anti-myosin light chain phosphatase target subunit 1 (p-MYPT1)-Thr696 (1:1000; Millipore, Tullagreen Carrigtwohill, Cork, Ireland, Cat# ABS45, RRID: AB_10562238), rabbit anti-iNOS (1:1000, Enzo Life Sciences, Cat# ADI-KAS-NO001, RRID: AB_10622409), mouse anti-Arg-1 (1:1000, BD Biosciences, Cat# 610708, RRID: AB_398031), rabbit anti-toll like receptor 2 (TLR-2) (1:1000; Cell Signaling Technology, Cat# 2229, RRID: AB_2204442), rabbit anti-TLR-4 (1:1000, Cell Signaling Technology, Cat# 14358, RRID: AB_2798460), rabbit anti-myeloid differentiation factor 88 (Myd88) (1:1000; Abcam, Cambridge, UK; Cat# ab2064, RRID: AB_302807), rabbit anti-p-Jun N-terminal kinase (p-JNK) (1:1000, Cell Signaling Technology, Cat# 4668, RRID: AB_823588), mouse anti-p-extracellular regulated protein kinases (p-ERK) (1:1000; Santa Cruz Biotechnology, Dallas, TX, USA, Cat# sc-81492, RRID: AB_1125801), mouse anti-p-P38 (1:1000; Santa Cruz Biotechnology, Cat# sc-7973, RRID: AB_670359), rabbit anti-p-nuclear factor kappa B (p-NF-κB) (1:1000, Cell Signaling Technology, Cat# 3037, RRID: AB_2341216), rabbit anti-ribosomal protein S3 (RPS3) (1:1000, Abcam, Cat# ab128995, RRID: AB_11145466), anti-GAPDH (1:10,000, Cell Signaling Technology, Cat# 2118, RRID: AB_561053), anti-mouse IgG HRP-linked secondary antibody (1:10,000, Cell Signaling Technology, Cat# 7076, RRID: AB_330924), and anti-rabbit IgG HRP-linked secondary antibody (1:10,000, Cell Signaling Technology, Cat# 7074, RRID: AB_2099233).

Techniques: In Vitro, Injection, Isolation, Magnetic Beads, Labeling, Flow Cytometry, Fluorescence

Journal: Neural Regeneration Research

Article Title: Fasudil-modified macrophages reduce inflammation and regulate the immune response in experimental autoimmune encephalomyelitis

doi: 10.4103/1673-5374.379050

Figure Lengend Snippet: Effects of Fasudil on iNOS/NO and Arg-1 expression. A mouse model of chronic EAE was induced by subcutaneous injection of myelin oligodendrocyte glycoprotein peptde 35–55. Macrophages from EAE mice isolated with magnetic beads were treated with 15 μg/mL of Fasudil at 37°C in 5% CO 2 atmosphere for 72 hours. (A) Arg-1 and iNOS expression levels were determined by western blot. (B) The Griess technique was used to determine the NO content of the supernatant, and Arg-1 activity was determined using an Arg-1 assay kit. Fasudil reduced iNOS/NO production by and enhanced Arg-1 release from macrophages. Quantitative data are shown as the mean ± SEM of four to six exemplars from each group. ** P < 0.01, *** P < 0.001 (Student’s t -test). Arg-1: Arginase-1; EAE: experimental autoimmune encephalomyelits; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; iNOS: inducible nitric oxide synthase.

Article Snippet: The antibodies used for western blotting were as follows: mouse anti-ROCK-II (1:1000; BD Biosciences, Cat# 610624, RRID: AB_397956), rabbit anti-myosin light chain phosphatase target subunit 1 (p-MYPT1)-Thr696 (1:1000; Millipore, Tullagreen Carrigtwohill, Cork, Ireland, Cat# ABS45, RRID: AB_10562238), rabbit anti-iNOS (1:1000, Enzo Life Sciences, Cat# ADI-KAS-NO001, RRID: AB_10622409), mouse anti-Arg-1 (1:1000, BD Biosciences, Cat# 610708, RRID: AB_398031), rabbit anti-toll like receptor 2 (TLR-2) (1:1000; Cell Signaling Technology, Cat# 2229, RRID: AB_2204442), rabbit anti-TLR-4 (1:1000, Cell Signaling Technology, Cat# 14358, RRID: AB_2798460), rabbit anti-myeloid differentiation factor 88 (Myd88) (1:1000; Abcam, Cambridge, UK; Cat# ab2064, RRID: AB_302807), rabbit anti-p-Jun N-terminal kinase (p-JNK) (1:1000, Cell Signaling Technology, Cat# 4668, RRID: AB_823588), mouse anti-p-extracellular regulated protein kinases (p-ERK) (1:1000; Santa Cruz Biotechnology, Dallas, TX, USA, Cat# sc-81492, RRID: AB_1125801), mouse anti-p-P38 (1:1000; Santa Cruz Biotechnology, Cat# sc-7973, RRID: AB_670359), rabbit anti-p-nuclear factor kappa B (p-NF-κB) (1:1000, Cell Signaling Technology, Cat# 3037, RRID: AB_2341216), rabbit anti-ribosomal protein S3 (RPS3) (1:1000, Abcam, Cat# ab128995, RRID: AB_11145466), anti-GAPDH (1:10,000, Cell Signaling Technology, Cat# 2118, RRID: AB_561053), anti-mouse IgG HRP-linked secondary antibody (1:10,000, Cell Signaling Technology, Cat# 7076, RRID: AB_330924), and anti-rabbit IgG HRP-linked secondary antibody (1:10,000, Cell Signaling Technology, Cat# 7074, RRID: AB_2099233).

Techniques: Expressing, Injection, Isolation, Magnetic Beads, Western Blot, Activity Assay