Journal: bioRxiv

Article Title: SARS-CoV-2 infection is associated with intestinal permeability, systemic inflammation, and microbial dysbiosis in hospitalized COVID-19 patients

doi: 10.1101/2023.12.07.570670

Figure Lengend Snippet: Plasma concentrations for I-FABP ( A ), Zonulin ( B ), LBP ( C ), and sCD14 ( D ) measured by ELISA and circulating fatty acids propionic acid ( E ), decanoic acid ( F ), butyric acid ( G ), nonanoic acid ( H ), and isovaleric acid ( I ) measured by LC-MS/MS. Comparisons between groups were performed by Kruskal-Wallis tests, followed by Dunn post-hoc tests if adjusted p values were below 0.05. Pairwise comparisons between each variable were corrected separately for false discovery rate by the Benjamini-Hochberg method and adjusted p values <0.05 were considered significant.

Article Snippet: Gut barrier damage biomarkers, including LBP (cell sciences CKH113), sCD14 (R&D Systems QK383), zonulin (MyBioSource MBS706368), and I-FABP (R&D Systems DFBP20) were measured using commercially available assays according to manufacturer’s guidelines.

Techniques: Clinical Proteomics, Enzyme-linked Immunosorbent Assay, Liquid Chromatography with Mass Spectroscopy

Journal: Scientific reports

Article Title: Blood-brain barrier and gut barrier dysfunction in chronic kidney disease with a focus on circulating biomarkers and tight junction proteins.

doi: 10.1038/s41598-022-08387-7

Figure Lengend Snippet: Figure 2. Analysis of soluble CD14 (sCD14) levels in haemodialysis (HD) and non-CKD controls (control). (A) Soluble CD14 levels. The number of samples (n) that each analyte was detected in is noted for each group. Data presented at median and interquartile range. Statistical significance, ***p < 0.001, ****p < 0.0001. (B) Spearman’s correlation between sCD14 and trimethylamine N-oxide (TMAO) levels.

Article Snippet: The S100B ELISA kit was performed in accordance with manufacturer guidelines, with the alteration that samples were undiluted. sCD14 levels in HD patients was previously recorded and reported87, for measurements in non-CKD controls the same CD14 ELISA kit (DC140, R&D Systems, UK) was used, and run in accordance with manufacturer guidelines.

Techniques: Control

Journal: Scientific reports

Article Title: Blood-brain barrier and gut barrier dysfunction in chronic kidney disease with a focus on circulating biomarkers and tight junction proteins.

doi: 10.1038/s41598-022-08387-7

Figure Lengend Snippet: Figure 1. Analysis of serum biomarkers in haemodialysis (HD) and non-CKD controls (control). (A) Brain-derived neurotrophic factor (BDNF), (B) Neuron-specific enolase (NSE) were measured by enzyme- linked immunosorbent assay (ELISA). (C) Trimethylamine N-oxide (TMAO) was measured by liquid chromatography-mass spectrometry (LC–MS). The number of samples (n) that each analyte was detected in is noted for each group. Data presented at median and interquartile range. Statistical significance, ***p < 0.001, ****p < 0.0001. (D) Spearman’s correlation between BDNF and TMAO levels. (E) Spearman’s correlation between NSE and TMAO levels.

Article Snippet: The S100B ELISA kit was performed in accordance with manufacturer guidelines, with the alteration that samples were undiluted. sCD14 levels in HD patients was previously recorded and reported87, for measurements in non-CKD controls the same CD14 ELISA kit (DC140, R&D Systems, UK) was used, and run in accordance with manufacturer guidelines.

Techniques: Control, Derivative Assay, Enzyme-linked Immunosorbent Assay, Liquid Chromatography, Mass Spectrometry, Liquid Chromatography with Mass Spectroscopy

Journal: Annals of Gastroenterology : Quarterly Publication of the Hellenic Society of Gastroenterology

Article Title: Systemic levels of human β-defensin 1 are elevated in patients with cirrhosis

doi:

Figure Lengend Snippet: Elevated soluble CD14 (sCD14) concentration in the serum of cirrhotic patients. Concentration of sCD14 was measured in healthy controls (n=31), patients with chronic viral hepatitis (n=26) and patients with cirrhosis (n=50) as described in patients and methods. Data is presented graphically as Whisker box-plots

Article Snippet: For sCD14, human CD14 ELISA Development kit (R&D Systems, Abingdon, UK) was used following manufacturer’s instructions.

Techniques: Concentration Assay, Whisker Assay

Journal: Annals of Gastroenterology : Quarterly Publication of the Hellenic Society of Gastroenterology

Article Title: Systemic levels of human β-defensin 1 are elevated in patients with cirrhosis

doi:

Figure Lengend Snippet: High correlation between the levels of human beta defensin-1 (hBD-1) and soluble CD14 (sCD14) in the hepatic veins of cirrhotic patients . Concentrations of hBD-1 and sCD14 were measured as described in patients and methods. Each dot corresponds to individual patients with cirrhosis (n=45). Analysis was performed in samples collected from peripheral veins (n=25, ) and from hepatic veins (n=20, )

Article Snippet: For sCD14, human CD14 ELISA Development kit (R&D Systems, Abingdon, UK) was used following manufacturer’s instructions.

Techniques:

Journal: Annals of Gastroenterology : Quarterly Publication of the Hellenic Society of Gastroenterology

Article Title: Systemic levels of human β-defensin 1 are elevated in patients with cirrhosis

doi:

Figure Lengend Snippet: Soluble CD14 (sCD14) and lipopolysaccharide binding protein (LBP) strongly correlate in serum of patients with cirrhosis. Concentrations of sCD14 were measured as described in Patients and Methods. Concentrations of LBP were measured by a commercially available ELISA, according to manufacturer’s instructions. Each dot corresponds to individual patients with cirrhosis (n=36). Analysis was performed in samples collected from peripheral veins

Article Snippet: For sCD14, human CD14 ELISA Development kit (R&D Systems, Abingdon, UK) was used following manufacturer’s instructions.

Techniques: Binding Assay, Enzyme-linked Immunosorbent Assay

Journal: Nature Microbiology

Article Title: Vaccine plus microbicide effective in preventing vaginal SIV transmission in macaques

doi: 10.1038/s41564-023-01353-7

Figure Lengend Snippet: a , Comparison of SAMT-247 non-treated/treated effector cell-mediated ADCC activity in the vaccine ( n = 18) and vaccine + SAMT-247 groups ( n = 20; P < 0.0001). b , Correlation of SAMT-247-induced ADCC activity with number of intravaginal challenges in the vaccine + SAMT-247 group ( n = 20; P = 0.024). c , d , Intracellular Granzyme B, perforin, IFN‐γ and TNF-α in macaque rectal mucosal ( n = 9) NKG2A + cells in the presence or absence of different stimuli. e , Macaque rectal mucosal NKp44 + IL-17 + cells in the presence or absence of different stimuli ( n = 9). f , Correlation of efferocytosis with number of intravaginal challenges in animals in the vaccine group ( n = 18; P = 0.01). g , h , Comparison of percentage of efferocytosis ( P < 0.0001) ( g ) and efferocytosis MFI ( P < 0.0001) ( h ) using week 14 CD14 + monocytes in all vaccinated animals ( n = 38). i , Correlation of SAMT-247-induced efferocytosis (SAMT-247-untreated efferocytosis subtracted from SAMT-247-treated efferocytosis) with number of intravaginal challenges in the vaccine + SAMT-247 group ( n = 20; P = 0.065). Data shown in a , c , d , e , g and h were analysed with the two-tailed Wilcoxon signed-rank test. Data shown in b , f and i were analysed with the two-tailed Spearman correlation test. Horizontal and vertical bars denote mean and standard deviation, respectively.

Article Snippet: CD14 + cells were isolated from cryopreserved PBMCs (10 × 10 6 cells) collected following pre-study and 2 weeks post last immunization (week 14) by using non-human primate CD14 MicroBeads (#130-091-097, Miltenyi Biotec) following manufacturer instructions.

Techniques: Comparison, Activity Assay, Two Tailed Test, Standard Deviation

Journal: Nature Microbiology

Article Title: Vaccine plus microbicide effective in preventing vaginal SIV transmission in macaques

doi: 10.1038/s41564-023-01353-7

Figure Lengend Snippet: a, b) Intracellular Granzyme B, perforin, IFN‐γ, and TNF-α in healthy human (n = 6) blood NKG2A + cells in the presence or absence of different stimuli. c ) Comparison of Env-specific rectal NKp44 + IL-17 + cells between vaccine+SAMT-247 (n = 20) and vaccine group (n = 18) 1 week post last vaccination ( P = 0.43). d ) Correlation of rectal mucosal Env-specific NKp44 + IL-17 + cells with number of intra-vaginal challenges in the vaccine group (n = 18). e ) Gating of NKG2A + NK cells, NKp44 + ILCs, and NKG2A – NKp44 – ILCs in rectal mucosal samples in the presence of PMA or PMA + SAMT-247 at 12 hours post stimulation. Gating was done on singlets, live, CD45 + , CD3 − , CD20 − , CD11b − cells. f ) Gating of NKp44 + IL-17 + ILCs in the rectal mucosal sample in the presence of PMA or PMA + SAMT-247 at 12 hours post stimulation. g ) Correlation of efferocytosis percentage with number of intra-vaginal challenges in the vaccine+SAMT-247 group (n = 20). ( h-i ) Comparison of h ) percentage of efferocytosis ( P < 0.0001) and i ) efferocytosis MFI ( P < 0.0001) using pre CD14 + monocytes in all vaccinated animals (n = 38). Data shown in ( a, b, h, i ) were analyzed with the two-tailed Wilcoxon signed-rank test or two-tailed Mann-Whitney test. Data shown in ( d, g ) were analyzed with the two-tailed Spearman correlation test.

Article Snippet: CD14 + cells were isolated from cryopreserved PBMCs (10 × 10 6 cells) collected following pre-study and 2 weeks post last immunization (week 14) by using non-human primate CD14 MicroBeads (#130-091-097, Miltenyi Biotec) following manufacturer instructions.

Techniques: Comparison, Two Tailed Test, MANN-WHITNEY

Journal: Nature Microbiology

Article Title: Vaccine plus microbicide effective in preventing vaginal SIV transmission in macaques

doi: 10.1038/s41564-023-01353-7

Figure Lengend Snippet: a , Representative imaging of human NKG2A + cells unstimulated or stimulated with SAMT-247, PMA or PMA + SAMT-247. b , Mean zinc intensity in NKG2A + cells of the healthy human donor in the presence or absence of zinc chelator in different stimulation conditions ( n = 8). Fluorescence intensity of each field was measured for zinc expression as indicated by green colour, and the total number of DAPI positive cells were counted to determine the mean intensity of zinc/cells using iMARIS software. The mean of two duplicate fields was evaluated for the calculation. c , Comparison of expressions of NKG2A marker in macaques in the absence or presence of zinc chelator and stimuli in the vaccine + SAMT-247 group ( n = 4) and vaccine group ( n = 2). d – g , Comparison of expressions of granzyme B, perforin, IFN‐γ and TNF-α by macaque blood NKG2A + cells from week 17 in the absence or presence of different stimulations and zinc chelator in the vaccine + SAMT-247 group ( n = 4) and vaccine group ( n = 2). h , i , Evaluation of the frequency of CD14 + monocytes and CD14 + IL-10 + monocytes in the absence or presence of zinc chelator and stimuli in the vaccine + SAMT-247 group ( n = 4) and vaccine group ( n = 2). Data shown in b – i were analysed with the two-tailed Wilcoxon signed-rank test. Horizontal and vertical bars denote mean and standard deviation, respectively.

Article Snippet: CD14 + cells were isolated from cryopreserved PBMCs (10 × 10 6 cells) collected following pre-study and 2 weeks post last immunization (week 14) by using non-human primate CD14 MicroBeads (#130-091-097, Miltenyi Biotec) following manufacturer instructions.

Techniques: Imaging, Fluorescence, Expressing, Software, Comparison, Marker, Two Tailed Test, Standard Deviation

Journal: Nature Microbiology

Article Title: Vaccine plus microbicide effective in preventing vaginal SIV transmission in macaques

doi: 10.1038/s41564-023-01353-7

Figure Lengend Snippet: Vaccination-induced ADCC results in apoptosis of SIV-infected cells, which in turn are cleared by efferocytes to avoid inflammation and preserve tissue homeostasis. Vaccine-induced IL-10 expression in CD14 + monocytes further augments efferocytosis. Vaccine-induced NKp44 + cells produce the IL-17 cytokine that maintains mucosal epithelium integrity. All of these protective effector responses were enhanced dramatically in the vaccine + SAMT-247 group, increasing protection from SIV mac251 acquisition. The scheme is adapted from Bissa et al. .

Article Snippet: CD14 + cells were isolated from cryopreserved PBMCs (10 × 10 6 cells) collected following pre-study and 2 weeks post last immunization (week 14) by using non-human primate CD14 MicroBeads (#130-091-097, Miltenyi Biotec) following manufacturer instructions.

Techniques: Infection, Expressing

Journal: Frontiers in immunology

Article Title: The significance of CD16+ monocytes in the occurrence and development of chronic thromboembolic pulmonary hypertension: insights from single-cell RNA sequencing.

doi: 10.3389/fimmu.2024.1446710

Figure Lengend Snippet: FIGURE 2 Changes in peripheral blood monocyte subsets in CTEPH patients. (A) UMAP plot of Mononuclear Phagocyte System (MPS) was divided into three cell types: monocytes, macrophages, and conventional dendritic cells (cDCs). Subsequently, monocytes were further divided into CD14+ monocytes and CD16+ monocytes. (B) A dot plot was used to annotate marker genes for CD14+ monocytes and CD16+ monocytes, along with a heatmap displaying the top 10 differentially expressed genes between the two cell types. (C) Ro/e (ratio of observed cell number to expected cell number) revealed the proportion of CD16+ monocytes in the CTEPH-N, CTEPH-I and HC groups. (D) The heat map showed the degree of correlation, and the numbers in the graph represented the correlation coefficient. (6MWD: 6-minute walk distance; PVR: pulmonary vascular resistance) (E) Schematic diagram of the gating strategy for distinguishing CD14+ monocytes from CD16+ monocytes by flow cytometry. (F) Differences in the proportion of CD16+ monocyte subsets in total monocytes between CTEPD patients (n = 15) and matched healthy controls (n = 15) were assessed by flow cytometry. All data were presented as means ± SEM, ***P < 0.001.

Article Snippet: Then the cells were resuspend in the flow cytometer wash buffer (2% FBS in PBS) and stained with the following antibodies according to the standard protocol: CD14-PE (Elabscience, E-ABF1209D) and CD16-FITC (Elabscience, E-AB-F1236C) for monocyte labeling.

Techniques: Marker, Cytometry

Journal: Frontiers in immunology

Article Title: The significance of CD16+ monocytes in the occurrence and development of chronic thromboembolic pulmonary hypertension: insights from single-cell RNA sequencing.

doi: 10.3389/fimmu.2024.1446710

Figure Lengend Snippet: FIGURE 3 The functional characteristics of peripheral blood CD16+ monocytes in CTEPH patients. (A) GO analysis (biological process) of upregulated genes in CD16+ monocytes compared with CD14+ monocytes in CTEPH patients. (B) KEGG analysis of upregulated genes in CD16+ monocytes compared with CD14+ monocytes in CTEPH patients. (C) GSEA bar plot of CD16+ monocytes versus CD14+ monocytes in patients with CTEPH. (D) GO analysis (biological process) of upregulated genes in CD16+ monocytes between the CTEPH and healthy control samples. (E) KEGG analysis of upregulated genes in CD16+ monocytes between CTEPH patients and healthy controls. (F) GSVA heatmap of CD16+ monocytes between CTEPH patients and healthy controls. (G) Heat map of transcription factors upregulated in CD16+ monocytes between CTEPH patients and healthy controls.

Article Snippet: Then the cells were resuspend in the flow cytometer wash buffer (2% FBS in PBS) and stained with the following antibodies according to the standard protocol: CD14-PE (Elabscience, E-ABF1209D) and CD16-FITC (Elabscience, E-AB-F1236C) for monocyte labeling.

Techniques: Functional Assay, Control

Journal: bioRxiv

Article Title: UBA1 Mutations Drive RIPK1-Mediated Cell Death and Monocyte Dysfunction in VEXAS Syndrome

doi: 10.1101/2025.10.06.680650

Figure Lengend Snippet: Regulated cell death pathways are activated in myeloid cells from VEXAS patients. ( A ) Leucocytes, neutrophils, monocytes, lymphocytes count from patients with VEXAS and elderly gender-matched healthy controls (HC). Panels show individual data (dots) and means ± SEM (histograms). P values were determined by the Mann-Whitney test. ( B ) Hematoxylin-eosin staining of UBA1-mutated Sweet-like lesion revealing karyorrhectic nuclei and apoptotic debris in VEXAS (arrows). Illustrative picture is shown (Magnification x10 and x40, scale bar 100 μm and 50µm). ( C ) Representative immunofluorescence images of pMLKL (green) staining on CD14+ sorted cells form 3 active VEXAS patients and 3 elderly gender-matched HC. Nuclei are stained in blue with Hoechst. Percentage of pMLKL cell surface area per CD14+ cells, data are shown and means (Histograms) ± SEM. Forty cells were quantified for each patient. ( D ) Multiplex Immunofluorescence of skin biopsy samples from VEXAS skin lesion showing the co-expression of cleaved GSDMD, MLKL, cleaved caspase-3 and phosphorylated RIPK1 within CD68+ infiltrates in VEXAS. ( E ) Gene set enrichment analysis of apoptosis (KEGG), necroptosis (GOBP) and pyroptosis (GOBP) pathways enriched in VEXAS lesional skin (n=6) versus non lesional skin from HC (n=5) adapted form from dataset GSE245639. *P□<□0.05; **P□<□0.01; ***P□<□0.001, ****P□<□0.0001. SEM, HC, Healthy Control; Standard Error of the Mean; VEXAS, Vacuoles, E1 enzyme, X-linked, Autoinflammatory, Somatic; WT, Wild-Type.

Article Snippet: For CD14+ cells form patients, cells were sorted from 2mL fresh Whole Blood collected in EDTA tube and sorted using StraightFrom® Whole Blood CD14 MicroBeads and Whole Blood column kit (Milteny Biotec, #130-090-879) according to the manufacturer’s instructions.

Techniques: MANN-WHITNEY, Staining, Immunofluorescence, Multiplex Assay, Expressing, Control

Journal:

Article Title: LPS-induced IL-6, IL-8, VCAM-1, and ICAM-1 Expression in Human Lymphatic Endothelium

doi: 10.1369/jhc.7A7299.2007

Figure Lengend Snippet: Expression of toll-like receptor 4 (TLR4), MD-2, and CD14. (A) Immunohistochemical analysis. The cell membrane was visualized with rhodamine-conjugated Con A (Con A, in red), and the reaction products to antibodies for TLR4, MD-2, and CD14 were visualized with an AF488-conjugated second antibody (AF488, in green). Merged images indicate that the expression of TLR4, MD-2, and CD14 was immunohistochemically observed in the cytoplasm and on the cell membrane of LEC, and that the three were detected on the membrane of HEK-293 cells stably transfected with the human TLR4, MD-2, and CD14 genes (tHEK), and not in the negative control HEK-293 cells (HEK). Bar = 100 μm. (B) RT-PCR analysis. RT-PCR products for TLR4, MD-2, and CD14 mRNAs were not detected in HEK-293 but were detected in LEC and in tHEK. MW, molecular weight marker.

Article Snippet: The cells were immunostained with 1 μg/ml of rabbit antiserum to human Prox1 (AngioBio Co.; Del Mar, CA) and of monoclonal antibodies to human podoplanin (AngioBio), VCAM-1, and ICAM-1 (R and D Systems, Inc.; Minneapolis, MN), and also immunostained with 5 μg/ml of monoclonal antibodies to TLR4, MD-2, and CD14 (R and D Systems).

Techniques: Expressing, Immunohistochemical staining, Membrane, Stable Transfection, Transfection, Negative Control, Reverse Transcription Polymerase Chain Reaction, Molecular Weight, Marker

Journal:

Article Title: LPS-induced IL-6, IL-8, VCAM-1, and ICAM-1 Expression in Human Lymphatic Endothelium

doi: 10.1369/jhc.7A7299.2007

Figure Lengend Snippet: Expression of genes for lymphatic endothelial markers, and TLR-associated and leukocyte adhesion molecules in human neonatal dermal lymphatic microvascular endothelial cells with LPS treatments

Article Snippet: The cells were immunostained with 1 μg/ml of rabbit antiserum to human Prox1 (AngioBio Co.; Del Mar, CA) and of monoclonal antibodies to human podoplanin (AngioBio), VCAM-1, and ICAM-1 (R and D Systems, Inc.; Minneapolis, MN), and also immunostained with 5 μg/ml of monoclonal antibodies to TLR4, MD-2, and CD14 (R and D Systems).

Techniques: Expressing, Control, Marker, Activation Assay

Journal: Frontiers in Immunology

Article Title: PFKFB3 overexpression in monocytes of patients with colon but not rectal cancer programs pro-tumor macrophages and is indicative for higher risk of tumor relapse

doi: 10.3389/fimmu.2022.1080501

Figure Lengend Snippet: Monocyte subpopulations` content in healthy individuals and CRC patients.

Article Snippet: For immunofluorescence (IF) staining, tumor FFPE clinical samples were treated with xylol solution and blocked with 3% BSA in PBS for 45 min, incubated with a combination of primary antibodies for 1,5 h; washed, and incubated with a combination of appropriate secondary antibodies for 45 min. Anti-PFKFB3 rabbit monоclonal antibody (1:50, #ab181861, Abcam, USA); anti-CD68 monoclonal mouse antibody (1:100, #NBP2-44539, clone KP1, Novus Biologicals); anti-CD14 polyclonal sheep antibody (1:50, #BAF383, R&D Systems) were used.

Techniques:

Journal: Frontiers in Immunology

Article Title: PFKFB3 overexpression in monocytes of patients with colon but not rectal cancer programs pro-tumor macrophages and is indicative for higher risk of tumor relapse

doi: 10.3389/fimmu.2022.1080501

Figure Lengend Snippet: The distribution of CD163+ and CCR2+ peripheral blood monocytes in patients with colon and rectal cancers. Individual profiles of CCR2+ and CD163+ monocyte subsets for each patient with rectal and colon cancers. (A) , The distribution of monocytes of classical (CD14+CD16-), intermediate (CD14+CD16+) and non-classical (CD14-CD16+) populations expressing CCR2 (upper panel) and CD163 (lower panel) is demonstrated before and after NAC and after surgical resection in rectal cancer patients. (B) , The distribution of monocytes of classical (CD14+CD16-), intermediate (CD14+CD16+) and non-classical (CD14-CD16+) populations expressing CCR2 (upper panel) and CD163 (lower panel) is demonstrated before and after surgical resection in colon cancer patients. (C) , Associations of CCR2-expressing monocyte subsets with hematogenous and lymphatic metastasis in rectal cancer patients. (D) , Associations of CD163-expressing monocyte subsets with hematogenous and lymphatic metastasis in colon cancer patients. M 0 , metastasis-negative status, M 1 , metastasis-positive status. N 0 , lymph node-negative status, N 1-3 , lymph node-positive status.

Article Snippet: For immunofluorescence (IF) staining, tumor FFPE clinical samples were treated with xylol solution and blocked with 3% BSA in PBS for 45 min, incubated with a combination of primary antibodies for 1,5 h; washed, and incubated with a combination of appropriate secondary antibodies for 45 min. Anti-PFKFB3 rabbit monоclonal antibody (1:50, #ab181861, Abcam, USA); anti-CD68 monoclonal mouse antibody (1:100, #NBP2-44539, clone KP1, Novus Biologicals); anti-CD14 polyclonal sheep antibody (1:50, #BAF383, R&D Systems) were used.

Techniques: Expressing

Journal: Frontiers in Immunology

Article Title: PFKFB3 overexpression in monocytes of patients with colon but not rectal cancer programs pro-tumor macrophages and is indicative for higher risk of tumor relapse

doi: 10.3389/fimmu.2022.1080501

Figure Lengend Snippet: An activator of glycolysis PFKFB3 is overexpressed in colon cancer and is indicative for higher risk of tumor relapse in colon cancer but not rectal cancer. (A) , Сolon cancer tissue is massively infiltrated by PFKFB3-positive monocytes. IF/confocal microscopy analysis was performed for 10 colon tumor tissues. The infiltration of CD14+CD68+PFKFB3+ cells was found in all samples. Representative image is given from one patient. Scale bar corresponds to 50 µm in main image and 20 µm in zoom image. (B) , Spearman correlation coefficients between PFKFB3 expression, M2 macrophage gene expressions and predicted cell abundance scores, FDR<0.05. (C) , Predicted cell composition of CD45+ AOIs and hierarchical clustering of AOIs. (D) , Difference in monocyte and macrophage cell abundance scores between the CD45+ AOIs in colon and rectal cancers (the Mann-Whitney U test was applied). (E) , PFKFB3 gene expression is elevated in patients with recurrence and larger tumor size in colon cancer. Variance in PFKFB3 expression was stabilized via the variance stabilizing transformation (VST). (F) , PFKFB3 had prognostic significance for the DFS and OS. High-risk group had worse survival rates compared to low-risk group. ROC analysis and Kaplan–Meier method were applied.

Article Snippet: For immunofluorescence (IF) staining, tumor FFPE clinical samples were treated with xylol solution and blocked with 3% BSA in PBS for 45 min, incubated with a combination of primary antibodies for 1,5 h; washed, and incubated with a combination of appropriate secondary antibodies for 45 min. Anti-PFKFB3 rabbit monоclonal antibody (1:50, #ab181861, Abcam, USA); anti-CD68 monoclonal mouse antibody (1:100, #NBP2-44539, clone KP1, Novus Biologicals); anti-CD14 polyclonal sheep antibody (1:50, #BAF383, R&D Systems) were used.

Techniques: Confocal Microscopy, Expressing, MANN-WHITNEY, Gene Expression, Transformation Assay