Journal: Cancers

Article Title: EGFR Mutation-Harboring Lung Cancer Cells Produce CLEC11A with Endothelial Trophic and Tumor-Promoting Activities

doi: 10.3390/cancers14051356

Figure Lengend Snippet: CLEC11A expression is increased in lung cancer cell lines expressing mutated EGFR. ( A ) H1299 cell derivatives were examined for CLEC11A mRNA expression using conventional RT-PCR and quantitative PCR assays. Levels of EGFR and phosphor-EGFR were detected by immune-blotting using specific antibodies. ( B ) NSCLC cell lines with endogenous wild type or mutated EGFR were examined for the indicated proteins by immune-blotting. The asterisk indicates a cross-reactive protein recognized by the antibody. ( C ) H1975, H1650, and HCC827 cells were treated with or without 1 mM of gefitinib for 2 days and the levels of indicated proteins were examined by immune-blotting.

Article Snippet: The slides were rinsed twice with PBS and then incubated with an anti-CLEC11A monoclonal antibody (MAB1904; R&D System) at 1:50 dilution for 2 h at room temperature.

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Real-time Polymerase Chain Reaction

Journal: Cancers

Article Title: EGFR Mutation-Harboring Lung Cancer Cells Produce CLEC11A with Endothelial Trophic and Tumor-Promoting Activities

doi: 10.3390/cancers14051356

Figure Lengend Snippet: CLEC11A expression is frequently detected in LAC tissues with an EGFR mutation. Tissue sections from LAC with or without EGFR kinase domain mutations were examined by IHC staining using an anti-CLEC11A monoclonal antibody. The light staining patterns of the connective tissue within the tumor sections served as internal, negative controls. Scale bar: 0.2 mm.

Article Snippet: The slides were rinsed twice with PBS and then incubated with an anti-CLEC11A monoclonal antibody (MAB1904; R&D System) at 1:50 dilution for 2 h at room temperature.

Techniques: Expressing, Mutagenesis, Immunohistochemistry, Staining

Journal: Cancers

Article Title: EGFR Mutation-Harboring Lung Cancer Cells Produce CLEC11A with Endothelial Trophic and Tumor-Promoting Activities

doi: 10.3390/cancers14051356

Figure Lengend Snippet: H1299 cells expressing CLEC11A have higher tumorigenic ability in vivo. ( A ) H1299-CLEC11A and CLEC11A (high) cells were established from permanently transfected cells. ( B ) H1299-Vec and H1299-CLEC11A cells were injected subcutaneously into nude mice. Tumors were harvested 5 weeks post-transplantation, weighed, and photographed. ( C ) Tumors formed by H1299-CLEC11A cells were significantly larger (Student t -test) than those formed by control cells. ( D ) H1299-Vec and H1299-CLEC11A cells were plated at 1 × 10 5 cells/well and the cell numbers were measured at indicated time points. ( E ) H1299-Vec and H1299-CLEC11A cells were plated at 100 cells/6 cm plate and cultured for 2 weeks and were fixed and stained with crystal violet. The numbers of colonies were determined and the statistical analyses were performed by the Student t -test.

Article Snippet: The slides were rinsed twice with PBS and then incubated with an anti-CLEC11A monoclonal antibody (MAB1904; R&D System) at 1:50 dilution for 2 h at room temperature.

Techniques: Expressing, In Vivo, Transfection, Injection, Transplantation Assay, Control, Cell Culture, Staining

Journal: Cancers

Article Title: EGFR Mutation-Harboring Lung Cancer Cells Produce CLEC11A with Endothelial Trophic and Tumor-Promoting Activities

doi: 10.3390/cancers14051356

Figure Lengend Snippet: Suppression of CLEC11A expression in HCC827 cells decreases tumorigenic ability. ( A ) HCC827-Vec and HCC827-shCLEC11A cells were established by permanent transfection of pSuper-puromycin and pSuper-shCLEC11A vectors, respectively. The expression levels of the indicated proteins in the conditioned medium (CM) were examined by immune-blotting. ( B ) HCC827-Vec and HCC827-shCLEC11A cells were injected subcutaneously into nude mice. Tumors were harvested 5 weeks post-transplantation, weighed, and photographed. ( C ) Tumors formed by HCC827-shCLEC11A cells were significantly larger (Student t -test) than those formed by control cells.

Article Snippet: The slides were rinsed twice with PBS and then incubated with an anti-CLEC11A monoclonal antibody (MAB1904; R&D System) at 1:50 dilution for 2 h at room temperature.

Techniques: Expressing, Transfection, Injection, Transplantation Assay, Control

Journal: Cancers

Article Title: EGFR Mutation-Harboring Lung Cancer Cells Produce CLEC11A with Endothelial Trophic and Tumor-Promoting Activities

doi: 10.3390/cancers14051356

Figure Lengend Snippet: CLEC11A-expressing tumors contain more CD31-positive cells of endothelial lineage. ( A ) Tissue sections prepared from tumors formed by H1299-Vec and H1299-CLEC11A cells were subjected to IHC staining using an anti-CD31 antibody. ( B ) Images from the experiments of panel A were quantitated and the data were analyzed by Student t -test. ( C ) In vivo angiogenesis assays were performed as described in Materials and Methods. The levels of blood vessel content in Matrigel with FGF1 or FGF1 plus CLEC11A were quantitated by measuring the hemoglobin (Hb) levels. The symbols (open and closed) with the same shape represented the data collected from Matrigel blobs of the same mouse. ( D ) This experiment was performed as described in panel C, except using VEGF-A instead of FGF1. The data were presented as the ratios of Hb content in the Matrigel blob containing VEGF plus CLEC11A over that containing VEGF alone from the same mouse.

Article Snippet: The slides were rinsed twice with PBS and then incubated with an anti-CLEC11A monoclonal antibody (MAB1904; R&D System) at 1:50 dilution for 2 h at room temperature.

Techniques: Expressing, Immunohistochemistry, In Vivo

Journal: Cancers

Article Title: EGFR Mutation-Harboring Lung Cancer Cells Produce CLEC11A with Endothelial Trophic and Tumor-Promoting Activities

doi: 10.3390/cancers14051356

Figure Lengend Snippet: CLEC11A is a factor with angiogenic potential. ( A ) The chemo-attractant ability of purified CLEC11A (30 ng/mL) was tested against SVEC4-10 cells in the presence or absence of the anti-CLEC11A monoclonal antibody (1 μg/mL) in the Transwell chambers. ( B ) The chemo-attractant ability of purified CLEC11A (30 ng/mL) was tested against primary AAE cells in the Transwell chambers. ( C ) Conditioned media of HCC827-Vec and HCC827-shCLEC11A cells were tested for their chemo-attractant ability against SVEC4-10 cells. ( D ) Tissue sections prepared from tumors formed by HCC827-Vec and HCC827-shCLEC11A cells were subjected to IHC staining using an anti-CD31 antibody. Staining images were quantitated and the data were analyzed by the Student t -test. ( E ) Overnight 1% serum-starved SVEC4-10 cells were treated with 30 ng/mL of CLEC11A for the indicated times and the levels proteins of interest were examined by immune-blotting. ( F ) The chemo-attractant activities of CLEC11A (30 ng/mL) or VEGF-A (20 ng/mL) were tested against SVEC4-10 cells in the presence or absence of vatalanib (5 mM) in the Transwell chambers. Statistical significances between treatments are presented as single asterisk ( p < 0.05) or double asterisks ( p < 0.01).

Article Snippet: The slides were rinsed twice with PBS and then incubated with an anti-CLEC11A monoclonal antibody (MAB1904; R&D System) at 1:50 dilution for 2 h at room temperature.

Techniques: Purification, Immunohistochemistry, Staining

Journal: Cancers

Article Title: EGFR Mutation-Harboring Lung Cancer Cells Produce CLEC11A with Endothelial Trophic and Tumor-Promoting Activities

doi: 10.3390/cancers14051356

Figure Lengend Snippet: Integrin receptors are involved in CLEC11A action on endothelial cells. ( A ) SVEC4-10 cells were plated on CLEC11A (2.5–10 μg/mL) or fibronectin (FN; 1 or 10 μg/mL)-coated plates for 20 min. The levels of indicated proteins were determined by immune-blotting. ( B ) Wild-type and different mutant CLEC11A proteins (30 ng/mL) were tested for their chemo-attractant ability against SVEC4-10 cells. ** p < 0.01. ( C ) His-tagged wild-type and different mutant CLEC11A proteins were immobilized on Co 2+ -conjugated agarose beads and were incubated with SVEC4-10 lysates for testing of their integrin-binding ability. The levels of indicated proteins in the complexes were determined by immune-blotting. ( D ) Overnight 1% serum-starved SVEC4-10 cells were treated with or without CLEC11A (30 ng/mL) for 20 min. The cells were then fixed and subjected to immuno-fluorescence staining using antibodies against pFAK (Y397) and active integrin β1 (ITGB1). DAPI was used to stain cell nuclei. Arrow heads indicate the signals of active ITGB1.

Article Snippet: The slides were rinsed twice with PBS and then incubated with an anti-CLEC11A monoclonal antibody (MAB1904; R&D System) at 1:50 dilution for 2 h at room temperature.

Techniques: Mutagenesis, Incubation, Binding Assay, Fluorescence, Staining

Journal: bioRxiv

Article Title: Distinct Microstructural Heterogeneities Underpin Specific Micromechanical Properties in Human ACL Femoral and Tibial Entheses

doi: 10.1101/2023.08.16.553628

Figure Lengend Snippet: Quantitative proteome analysis of the difference in expression of FE and TE interfaces compared to Ligament and SB. (A) Experimental procedure for the proteomic investigation. Volcano plots of univariate statistical analysis as applied to ligament-bone samples (n = 3 for each tissue). The plots are based on the fold change (log2) and the P-value (-log10) of all proteins identified in (B) proteins from FE and FL; (C) proteins from FE and FB; (D) proteins from TE and TL; (E) proteins from TE and TB. (F) Immunofluorescence staining of SLC24A1 (cyan) and CLEC11A (magenta) in ACL-femur tissues and SRPX (cyan) and MGP (magenta) in ACL-tibia tissues. Scale bar, 20 μm.

Article Snippet: Next, the FE samples were subjected to incubation with various primary antibodies, including SLC25A1 antibody (1:50, 15235-1-AP, Proteintech) and CLEC11A antibody (1:100, 55019-1-AP, Proteintech).

Techniques: Expressing, Immunofluorescence, Staining

Journal: Journal of Molecular Endocrinology

Article Title: CLEC11A improves insulin secretion and promotes cell proliferation in human beta-cells

doi: 10.1530/JME-22-0066

Figure Lengend Snippet: Expression of CLEC11A and its receptor ITGA11 in human islets and EndoC-βH1 cells. (A) Representative immunofluorescence staining of partially disrupted human islets revealed the expression and co-localization of CLEC11A (red) with insulin-secreting beta-cells (green) or with glucagon-secreting alpha-cells (green), respectively. Nuclei were stained with DAPI (blue). The negative control groups of disrupted human islets without primary antibodies were also shown. Scale bars, 50 μm. (B) Representative immunofluorescence staining of EndoC-βH1 cells. The staining of CLEC11A (red) and glucagon (green) was not detected in EndoC-βH1 cells. Scale bars, 50 μm. (C) Western blot and (D) RT-qPCR confirmed the expression of CLEC11A in human islets but not in EndoC-βH1 cells, whereas its receptor ITGA11 was found to be expressed in both human islets and EndoC-βH1 cells. The relative mRNA expression levels of CLEC11A and ITGA11 were first normalized by using the geometric average C t value of two endogenous controls GAPDH and ACTB , respectively, and further calculated with the 2 –△Ct method. Results are expressed as mean ± s.e.m. of n = 3 donors. *Indicates P < 0.05.

Article Snippet: Primary antibodies used for western blot were as follows: mouse anti-human SCGF/CLEC11A (1:500) (R&D Systems), rabbit anti-human ITGA11 (1:500) (LSBio, Shirley, MA, USA), rabbit anti-human PDX1 (1:3000) (Abcam), and rabbit anti-beta-actin (1:1000) (Cell Signaling Technology).

Techniques: Expressing, Immunofluorescence, Staining, Negative Control, Western Blot, Quantitative RT-PCR

Journal: Journal of Molecular Endocrinology

Article Title: CLEC11A improves insulin secretion and promotes cell proliferation in human beta-cells

doi: 10.1530/JME-22-0066

Figure Lengend Snippet: rhCLEC11A promotes beta-cell proliferation in human islets. Human islets (20–30 islets/treatment) were cultured in the absence or presence of different concentrations of rhCLEC11A indicated in the figures for 2 days and 7 days, respectively. (A) Representative image of immunofluorescence staining for EdU and insulin in human islets. EdU (green), insulin (red), and nuclei (blue) are shown. EdU-positive insulin-staining cells are indicated with arrows (white). Scale bars, 50 μm. The percentage of EdU-positive cells in insulin-staining beta-cells was calculated from the immunofluorescent images and expressed as fold control for (B) 2-day culture and (C) 7-day culture. (D) After 2-day and 7-day culture, the relative mRNA expression level of the PCNA gene from human islets was measured by RT-qPCR, normalized to endogenous control ACTB and calculated with the 2 –△△Ct method. Results are means ± s.e.m. of n = 3–4 donors from human islets. *Indicates P < 0.05.

Article Snippet: Primary antibodies used for western blot were as follows: mouse anti-human SCGF/CLEC11A (1:500) (R&D Systems), rabbit anti-human ITGA11 (1:500) (LSBio, Shirley, MA, USA), rabbit anti-human PDX1 (1:3000) (Abcam), and rabbit anti-beta-actin (1:1000) (Cell Signaling Technology).

Techniques: Cell Culture, Immunofluorescence, Staining, Control, Expressing, Quantitative RT-PCR

Journal: Journal of Molecular Endocrinology

Article Title: CLEC11A improves insulin secretion and promotes cell proliferation in human beta-cells

doi: 10.1530/JME-22-0066

Figure Lengend Snippet: rhCLEC11A induces the expression of transcription factors in human islets and EndoC-βH1 cells. The relative mRNA expression levels of genes related to insulin ( INS ) and transcription factors ( PDX1 and MAFA ) were measured by RT-qPCR, normalized to endogenous control ACTB (for human islets) and GAPDH (for EndoC-βH1 cells), and calculated with the 2 –△△Ct method, respectively. Respective gene expression in human islets with (A) 2-day culture in the absence or presence of 1000 ng/mL of rhCLEC11A and (B) 7-day culture in the absence or presence of 100 ng/mL of rhCLEC11A. (C) Respective gene expression in EndoC-βH1 cells cultured in the absence or presence of 10 ng/mL of rhCLEC11A for 2 days. (D) EndoC-βH1 cells were cultured in the absence or presence of different concentrations of rhCLEC11A (10 and 100 ng/mL, respectively) for 2 days. The expression level of PDX1 was confirmed by western blot, normalized to beta-actin and expressed as fold control. Results are expressed as means ± s.e.m. of n = 3–4 donors from human islets and means ± s.e.m. of n = 3–4 independent experiments from EndoC-βH1 cells. *indicates P < 0.05.

Article Snippet: Primary antibodies used for western blot were as follows: mouse anti-human SCGF/CLEC11A (1:500) (R&D Systems), rabbit anti-human ITGA11 (1:500) (LSBio, Shirley, MA, USA), rabbit anti-human PDX1 (1:3000) (Abcam), and rabbit anti-beta-actin (1:1000) (Cell Signaling Technology).

Techniques: Expressing, Quantitative RT-PCR, Control, Gene Expression, Cell Culture, Western Blot