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

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Tetraspanin CD9 negatively regulates lipopolysaccharide-induced macrophage activation and lung inflammation.

doi: 10.4049/jimmunol.0802797

Figure Lengend Snippet: FIGURE 4. The expression of CD14, complex formation of CD14 and TLR4, and their localization into the lipid raft are enhanced in CD9 KO BMDMs. A, BMDMs from WT and CD9 KO mice were stimulated with 1 g/ml LPS. After the indicated hours, cell lysates were electrophoresed on SDS-PAGE, transferred to a membrane, probed with anti-CD14 and anti-TLR4 Abs. Anti-actin blots show comparable amounts of protein loaded in each lane. B, WT and CD9 KO BMDMs were cultured in the absence () or presence () of LPS for 20 h. CD14 protein in whole cell lysates (WCL) and in immuno- precipitates with anti-TLR4 mAb (MTS510) was immunoblotted with biotinylated anti-CD14 Ab after SDS-PAGE. Anti-actin blots show comparable amounts of protein loaded in each lane. C, Cell lysates from WT and CD9 KO BMDMs cultured in the absence () or presence () of LPS for 2 h were centrifuged in sucrose density gradients. Fractions were collected from the top of the gradient and separated by SDS-PAGE. Protein distribution in the fractions was visualized by immunoblotting with anti-CD14, anti-TLR4, anti-CD9, anti-CD81, anti-CD45, and anti-flotillin-1 Abs. GM1 ganglioside was detected with HRP-conjugated cholera toxin by dot blot. The intensity of blots was quantified by densitometry. Percentage of density units of light membrane (LM) fractions (4 plus 5) were calculated and shown on the right of blots. D, Pooled LM fractions (4 and 5) and dense (D) fractions (9 and 10) from the sucrose gradients of WT cell lysate were subjected to imunoprecipiation with anti-CD9 mAb. After separation of the immunoprecipitates on SDS-PAGE, CD14 and CD9 were immunoblotted with biotinylated anti-CD14 Ab and anti-CD9 mAb, respectively. Data shown are from one representative of three similar experiments.

Article Snippet: Rat anti-CD14 mAb (rmC5–3) and goat biotinylated anti-CD14 polyclonal Ab (BAF9892) were purchased from BD Biosciences and R&D Systems, respectively.

Techniques: Expressing, SDS Page, Membrane, Cell Culture, Western Blot, Dot Blot

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Tetraspanin CD9 negatively regulates lipopolysaccharide-induced macrophage activation and lung inflammation.

doi: 10.4049/jimmunol.0802797

Figure Lengend Snippet: FIGURE 5. Accelerated IB degradation and analysis of other signaling molecules in LPS-activated CD9 KO BMDMs. A, BMDMs from WT and CD9 KO mice were stimulated with 1 g/ml LPS. After the indicated minutes, cell lysates were separated by SDS-PAGE, transferred to a mem- brane, and immunoblotted with anti-IB, anti-phosphorylated p38 (p- p38), and anti-p38 polyclonal Abs. Anti-actin blots show comparable amounts of protein loaded in each lane. B, SHP-1 was immunoprecipitated using anti-SHP-1 polyclonal Ab from cell lysates of WT and CD9 KO BMDMs stimulated with LPS for the indicated hours. After electrophoresis on SDS-PAGE and transfer to a membrane, tyrosine-phosphorylated SHP-1 was probed with anti-phosphotyrosine mAb (PY20), and the asso- ciated SIRP was blotted with anti-SIRP polyclonal Ab. SHP-1 and SIPR in whole cell lysates (WCL) were immunoblotted in parallel. Anti- actin blots show comparable amounts of protein loaded in each lane. Data shown are from one representative of three similar experiments.

Article Snippet: Rat anti-CD14 mAb (rmC5–3) and goat biotinylated anti-CD14 polyclonal Ab (BAF9892) were purchased from BD Biosciences and R&D Systems, respectively.

Techniques: SDS Page, Immunoprecipitation, Electrophoresis, Membrane