Journal: Frontiers in Oncology

Article Title: Characterization of Anti-Cancer Activities of Violacein: Actions on Tumor Cells and the Tumor Microenvironment

doi: 10.3389/fonc.2022.872223

Figure Lengend Snippet: The supernatant of violacein-killed tumor cells affects macrophage phenotype. HMDMs were differentiated and polarized in vitro into M0, M1, M2 (IL4), and M2 (IL10) macrophages for 24 h. (A) Differently polarized macrophages were imaged using the IncuCyte ® S3 system, and cells were grouped based on their eccentricity in an elongated or round morphology using the IncuCyte ® cell-by-cell analysis software. (B) Left: Representative pictures of M1 and M2 (IL10) macrophages are shown. Right: The violet mask labels the cells detected by the software as elongated. Scale bar = 200 µm. (C) Polarized macrophages were treated with dTCMs (37.5% v/v) from either HCT116, Huh7, or PANC-1 treated with either oxaliplatin (Oxa), violacein (Vio), or solvent control (co). Macrophage morphology was analyzed 48 h after treatment. n = 2 (triplicates). (D-F) M0 macrophages were treated with dTCMs (37.5% v/v) from Huh7 cells for 24 h. Gene expression was determined via qPCR and values were normalized to RNA18S as a housekeeping gene; 2 (triplicates). * p< 0.05, ** p< 0.01, *** p<0.001.

Article Snippet: Polarization of BMMs was performed in vitro : To obtain M1 polarization, media was supplemented with 20 ng/ml recombinant IFNγ (Biomol #87389.100) and 100 ng/ml LPS (Ultrapure LPS from Escherichia coli K12 #tlrl-peklps), while either 20 ng/ml IL4 (Miltenyi #130-094-061) or IL10 (Miltenyi #130-094-067) was used to obtain M2 polarization.

Techniques: In Vitro, Cell Analysis, Software, Solvent, Control, Gene Expression

Journal: Current Issues in Molecular Biology

Article Title: Transcriptomic Establishment of Pig Macrophage Polarization Signatures

doi: 10.3390/cimb45030151

Figure Lengend Snippet: Brief methods of bone marrow-derived macrophage induction.

Article Snippet: M2_IL4 + IL10 , M2 , 10 ng/mL IL10(Proteintech, USA, HZ-1145), and 10 ng/mL IL4 (Proteintech, Rosemount, IL, USA, HZ-1004).

Techniques:

Journal: Current Issues in Molecular Biology

Article Title: Transcriptomic Establishment of Pig Macrophage Polarization Signatures

doi: 10.3390/cimb45030151

Figure Lengend Snippet: Transcriptomic comparison of porcine macrophage phenotypes, M1 (M1_IFNγ + LPS, and M1_GM-CSF) versus M2 (M1_IFNγ + LPS, and M1_GM-CSF). ( A ) PCA clustering M1_IFNγ + LPS ( n = 4), M1_GM-CSF ( n = 4), M2_IL4 + IL10 ( n = 3), M2_M-CSF ( n = 3); ( B ) Top enriched GO biological processes of 730 DEG genes, M1 (M1_IFNγ + LPS, and M1_GM-CSF) and M2 (M2_IL4 + IL10, and M2_M-CSF); ( C ) Heatmap exhibiting 49 classical macrophage marker genes expression profile among M1_IFNγ + LPS, M1_GM-CSF, M2_IL4 + IL10, and M2_M-CSF; ( D ) The DEGs between M1 and M2 were studied by using the String online tool. The interaction between each protein pair is represented by lines, and the size of the circle is directly proportional to the degree.

Article Snippet: M2_IL4 + IL10 , M2 , 10 ng/mL IL10(Proteintech, USA, HZ-1145), and 10 ng/mL IL4 (Proteintech, Rosemount, IL, USA, HZ-1004).

Techniques: Comparison, Marker, Expressing

Journal: Current Issues in Molecular Biology

Article Title: Transcriptomic Establishment of Pig Macrophage Polarization Signatures

doi: 10.3390/cimb45030151

Figure Lengend Snippet: Comparison of transcriptomics in different phenotypes of porcine macrophages. ( A ) Heatmap showing that specific macrophage phenotypes were aggregated by the induction method, M1_IFNγ + LPS, and M1_GM-CSF; ( B ) Heatmap showing that specific macrophage phenotypes were aggregated by the induction method M2_IL4 + IL10 and M2_M-CSF; ( C ) GO pathway enrichment analysis of two polarized macrophages, M1_IFNγ + LPS and M1_GM-CSF; ( D ) GO pathway enrichment analysis of two polarized macrophages, M2_IL4 + IL10 and M2_M-CSF; ( E ) The DEGs were studied by using the String online tool, M1_IFNγ + LPS, and M1_GM-CSF. The interaction between each protein pair is represented by lines, and the size of the circle is directly proportional to the degree of interaction. We adopt a node number greater than 1; ( F ) The DEGs are studied by using the String online tool, M2_IL4 + IL10, and M2_M-CSF. The interaction between each protein pair is represented by lines, and the size of the circle is directly proportional to the degree of interaction. We adopt a node number greater than 2.

Article Snippet: M2_IL4 + IL10 , M2 , 10 ng/mL IL10(Proteintech, USA, HZ-1145), and 10 ng/mL IL4 (Proteintech, Rosemount, IL, USA, HZ-1004).

Techniques: Comparison

Journal: Current Issues in Molecular Biology

Article Title: Transcriptomic Establishment of Pig Macrophage Polarization Signatures

doi: 10.3390/cimb45030151

Figure Lengend Snippet: Validation of M1_IFNγ + LPS, M1_GMCSF, M2_M-CSF, and M2_IL4 + IL10 signatures. ( A ) SS2-infected macrophages were enriched in M1_IFNγ + LPS based on GSEA (Data source: Microarray analysis); ( B ) PRRSV-infected macrophages were enriched in M1_GM-CSF based on GSEA (Data source: RNA-seq); ( C ). T. gondii -infected macrophages were enriched in M2_IL4 + IL10 based on GSEA (Data source: RNA-seq); ( D ) T. gondii Me49 -infected macrophages were enriched in M2_M-CSF based on GSEA (Data source: RNA-seq).

Article Snippet: M2_IL4 + IL10 , M2 , 10 ng/mL IL10(Proteintech, USA, HZ-1145), and 10 ng/mL IL4 (Proteintech, Rosemount, IL, USA, HZ-1004).

Techniques: Biomarker Discovery, Infection, Microarray, RNA Sequencing

Journal: Frontiers in Oncology

Article Title: Characterization of Anti-Cancer Activities of Violacein: Actions on Tumor Cells and the Tumor Microenvironment

doi: 10.3389/fonc.2022.872223

Figure Lengend Snippet: The supernatant of violacein-killed tumor cells affects macrophage phenotype. HMDMs were differentiated and polarized in vitro into M0, M1, M2 (IL4), and M2 (IL10) macrophages for 24 h. (A) Differently polarized macrophages were imaged using the IncuCyte ® S3 system, and cells were grouped based on their eccentricity in an elongated or round morphology using the IncuCyte ® cell-by-cell analysis software. (B) Left: Representative pictures of M1 and M2 (IL10) macrophages are shown. Right: The violet mask labels the cells detected by the software as elongated. Scale bar = 200 µm. (C) Polarized macrophages were treated with dTCMs (37.5% v/v) from either HCT116, Huh7, or PANC-1 treated with either oxaliplatin (Oxa), violacein (Vio), or solvent control (co). Macrophage morphology was analyzed 48 h after treatment. n = 2 (triplicates). (D-F) M0 macrophages were treated with dTCMs (37.5% v/v) from Huh7 cells for 24 h. Gene expression was determined via qPCR and values were normalized to RNA18S as a housekeeping gene; 2 (triplicates). * p< 0.05, ** p< 0.01, *** p<0.001.

Article Snippet: Polarization of BMMs was performed in vitro : To obtain M1 polarization, media was supplemented with 20 ng/ml recombinant IFNγ (Biomol #87389.100) and 100 ng/ml LPS (Ultrapure LPS from Escherichia coli K12 #tlrl-peklps), while either 20 ng/ml IL4 (Miltenyi #130-094-061) or IL10 (Miltenyi #130-094-067) was used to obtain M2 polarization.

Techniques: In Vitro, Cell Analysis, Software, Solvent, Control, Gene Expression

Journal: Frontiers in Cell and Developmental Biology

Article Title: SLIT2 Overexpression in Periodontitis Intensifies Inflammation and Alveolar Bone Loss, Possibly via the Activation of MAPK Pathway

doi: 10.3389/fcell.2020.00593

Figure Lengend Snippet: Periodontitis-induced SLIT2 aggravates periodontitis and alveolar bone loss. Expression pattern of SLIT2 protein in, (A) Gingival tissue of periodontitis patients and healthy individuals ( n = 20), and (B) periodontal tissue of healthy and periodontitis wild-type or Slit2-Tg mice. (C) Serum level of SLIT2 protein in wild-type mice ( n = 5). (D) Micro-CT images of healthy and periodontitis-affected alveolar bone with the intact tooth of wild-type and Slit2-Tg mice. (E) Quantitative data of CEJ-ABC distance analyzed from micro-CT images. (F) H&E stained histological images of PAPT of wild-type and Slit2-Tg mice. (G) Quantitative data of alveolar bone loss around the periodontitis-affected tooth analyzed from histological images. Data are presented as mean ± SD ( n = 6). The significant difference among the groups, * P < 0.05, ** P < 0.01, *** P < 0.001. CEJ: cement-enamel junction, ABC: alveolar bone crest, Red dot line: CEJ level, Black dot line: ABC level, al: alveolar bone, de: dentine, blue arrow: inflammatory infiltration of the gingival epithelium, black arrow: alveolar bone resorption, double arrowhead (blue): CEJ-ABC distance, and WT: wild-type.

Article Snippet: However, the expression of M2 macrophage polarization related cytokines (such as Il10 and Il4 ) remained the same in Slit2-Tg PAPT and wild-type mice ( ).

Techniques: Expressing, Micro-CT, Staining

Journal: Frontiers in Cell and Developmental Biology

Article Title: SLIT2 Overexpression in Periodontitis Intensifies Inflammation and Alveolar Bone Loss, Possibly via the Activation of MAPK Pathway

doi: 10.3389/fcell.2020.00593

Figure Lengend Snippet: SLIT2 overexpression augments pathological changes in both periodontal milieu soft and bone tissues during periodontitis. (A) Representative histological images of PAPT sections showing TRAP+ osteoclasts. (B) Representative CD34 and CD45 immunohistochemistry images of PAPT sections. (C) Quantitative analysis of osteoclasts from TRAP-stained histological tissue sections ( n = 6). (D) Quantification of micro-vessel density from CD34 immunohistochemistry images ( n = 5). (E) Quantification of CD45 expressing cells from CD45 immunohistochemistry images ( n = 6). The significant difference among the groups, * P < 0.05, ** P < 0.01. Black arrow: TRAP+ multinucleated osteoclasts. Black triangle arrowhead: microvessel. WT: wild-type.

Article Snippet: However, the expression of M2 macrophage polarization related cytokines (such as Il10 and Il4 ) remained the same in Slit2-Tg PAPT and wild-type mice ( ).

Techniques: Over Expression, Immunohistochemistry, Staining, Expressing

Journal: Frontiers in Cell and Developmental Biology

Article Title: SLIT2 Overexpression in Periodontitis Intensifies Inflammation and Alveolar Bone Loss, Possibly via the Activation of MAPK Pathway

doi: 10.3389/fcell.2020.00593

Figure Lengend Snippet: SLIT2 overexpression triggers up-regulation of periodontal inflammatory factors. (A (i)) Relative mRNA expression, and (A (ii)) Heat map showing the differential expression pattern of inflammatory markers in PAPT analyzed by RNA sequencing ( n = 3). Relative mRNA expression of (B) Il6 , (C) Il1 β, and (D) Tnfα in periodontitis-affected tissue analyzed by RT-qPCR. Protein expression of, (E) IL-6, (F) IL-1β, and (G) TNF-α in periodontitis-affected tissue analyzed by ELISA. Data are presented as mean ± SD ( n = 6). Data are presented as mean ± SD. The significant difference among the groups, * P < 0.05, ** P < 0.01, *** P < 0.001. Red color intensity indicates upregulation, and blue color intensity indicates the downregulation of gene expression.

Article Snippet: However, the expression of M2 macrophage polarization related cytokines (such as Il10 and Il4 ) remained the same in Slit2-Tg PAPT and wild-type mice ( ).

Techniques: Over Expression, Expressing, Quantitative Proteomics, RNA Sequencing, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Gene Expression

Journal: Frontiers in Cell and Developmental Biology

Article Title: SLIT2 Overexpression in Periodontitis Intensifies Inflammation and Alveolar Bone Loss, Possibly via the Activation of MAPK Pathway

doi: 10.3389/fcell.2020.00593

Figure Lengend Snippet: SLIT2 overexpression induces M1 macrophage polarization in PAPT. (A (i)) Representative F4/80 immunohistochemistry images of the PAPT section. (A (ii)) Quantification of F4/80 expressing cells in a PAPT section from immunohistochemistry images ( n = 6). (B (i)) Flow cytometry analysis of F4/80 positive relative frequency of gated populations from PAPT. (B (ii)) Quantitative analysis of F4/80+ flow cytometry data ( n = 5). Relative mRNA expression of (C) M1, and (D) M2 macrophage polarization related genes analyzed by RNA sequencing ( n = 3). (E) Protein level expression of CD16/32 in PAPT ( n = 6). Protein level expression of, (F) CD16/32 in cell lysates of LPS-treated BMMs. Protein expression of (G) IL-6, (H) IL-1β, and (I) TNF-α in conditioned medium of LPS-treated BMMs ( n = 4). Data are presented as mean ± SD. The significant difference among the groups, * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: However, the expression of M2 macrophage polarization related cytokines (such as Il10 and Il4 ) remained the same in Slit2-Tg PAPT and wild-type mice ( ).

Techniques: Over Expression, Immunohistochemistry, Expressing, Flow Cytometry, RNA Sequencing

Journal: Frontiers in Cell and Developmental Biology

Article Title: SLIT2 Overexpression in Periodontitis Intensifies Inflammation and Alveolar Bone Loss, Possibly via the Activation of MAPK Pathway

doi: 10.3389/fcell.2020.00593

Figure Lengend Snippet: SLIT2/ROBO1 signaling possibly regulates the SLIT2-induced inflammation in periodontitis. (A (i)) Heat map the differential expression of Slit2 receptors Robo1-4 , and (A (ii)) Relative mRNA expression of Robo1 , Robo2 , Robo3 , and Robo4 in PAPT analyzed by RNA sequencing ( n = 3). Relative mRNA expression of, (B) Robo1 , (C) Robo2 , and (D) Robo4 in PAPT analyzed by RT-qPCR ( n = 6). (E) Heat map of differentially expressed 134 mRNAs in PAPT of Slit2-Tg mice to wild-type mice (fdr < 0.05,| FC| > 2). (F) GO pathway enrichment analysis. Data are presented as mean ± SD. The significant difference among the groups, * P < 0.05, ** P < 0.01.

Article Snippet: However, the expression of M2 macrophage polarization related cytokines (such as Il10 and Il4 ) remained the same in Slit2-Tg PAPT and wild-type mice ( ).

Techniques: Quantitative Proteomics, Expressing, RNA Sequencing, Quantitative RT-PCR

Journal: Frontiers in Cell and Developmental Biology

Article Title: SLIT2 Overexpression in Periodontitis Intensifies Inflammation and Alveolar Bone Loss, Possibly via the Activation of MAPK Pathway

doi: 10.3389/fcell.2020.00593

Figure Lengend Snippet: SLIT2 overexpression activates MAPK signaling pathway in periodontitis milieu. (A (i)) Relative mRNA expression ( n = 3), and (A (ii)) Heat map of the differential expression pattern of MAPK signaling pathway-related genes in PAPT analyzed by RNA sequencing. Relative mRNA expression of (B) Traf6 , and (C) p38 in PAPT analyzed by RT-qPCR ( n = 6). (D (i)) Representative western blot images of MAPK signaling related proteins. (D (ii), (iii)) Quantitative analysis of TRAP and P-P38 ( n = 4). Data are presented as mean ± SD. The significant difference among the groups, * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: However, the expression of M2 macrophage polarization related cytokines (such as Il10 and Il4 ) remained the same in Slit2-Tg PAPT and wild-type mice ( ).

Techniques: Over Expression, Expressing, Quantitative Proteomics, RNA Sequencing, Quantitative RT-PCR, Western Blot