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

Article Title: CCR7 signaling inhibits T cell proliferation.

doi: 10.4049/jimmunol.179.10.6485

Figure Lengend Snippet: FIGURE 2. CCR7 ligands suppress proliferation of both human and murine T cells. A, CCR7 human T cell line HUT78 cells were cultured in the presence of the indicated concentrations of CCL19-IgG, CCL19, or hIgG for 4 days. B, Human lymphocytes were isolated from peripheral blood and T cell separation was performed using negative selection by magnetic separation (MACS). Cells were stimulated with plate-bound anti-CD3 (-CD3; 3 g/ml) and anti-CD28 (-CD28; 1 g/ml) for 4 days in the presence of the indicated concentrations of recombinant CCL19, CCL19-IgG, or CCL21-IgG. Proliferation was measured by BrdU chemiluminescence. C, Murine T cells isolated from splenocytes of BALB/c mice were separated using negative selection by magnetic separation (MACS). Cells were stimulated with plate-bound anti-CD3 at the indicated concentrations. Cells were cultured with 1 g/ml anti-CD28 for 4 days in the presence or absence of CCL19-IgG, CCL21-IgG, or eotaxin (each 2.5 M) before proliferation was assessed using BrdU chemiluminescence. A–C, Mean proliferation SD from at least three experiments is shown; , p 0.05; p 0.01. D, Human T cells were fluorescence labeled with PKH26 and stimulated with immobilized anti-CD3/anti-CD28 for 4 days in the presence of 2.5 M CCL19-IgG or hIgG. Cell division was assessed by fluorescence fading using FACS analysis. The proportion of divided cells gated for CD3, CD4, or CD8 is indicated. The graph is representative of three repeated experiments with similar results. rlu, Relative luminescence unit.

Article Snippet: HUT78, a CCR7-expressing human T cell lymphoma cell line, and CHO cells were obtained from American Type Culture Collection.

Techniques: Cell Culture, Isolation, Selection, Recombinant, Labeling

Journal: Cancer Research

Article Title: Phage Display–Derived Human Monoclonal Antibodies Isolated by Binding to the Surface of Live Primary Breast Cancer Cells Recognize GRP78

doi: 10.1158/0008-5472.can-06-4686

Figure Lengend Snippet: Figure 1. Staining pattern of human mAbs Ab39 and Ab83 in human cancer biopsies and its comparison with anti-GRP78 staining as determined by immunohistochemical analysis. Ab39 (A), Ab83 (B), AbK2 (negative control; C), and rabbit anti-GRP78 antibody (H-129; D) were used to stain human cancer tissues, including lobular breast carcinoma (a), malignant melanoma (b), lung adenocarcinoma (c), and lung squamous cell carcinoma (d). Ab39, Ab83, and rabbit anti-GRP78 antibody stained cancer cells in all four specimens, whereas no staining was observed in adjacent normal tissue. No staining of cancer cells or adjacent normal tissue using control AbK2 was observed (C).

Article Snippet: Tissue sections were processed and stained with Ab39, as described in the immunohistochemistry section above, except that before incubation with Ab39, sections were preincubated with affinity-purified goat anti-GRP78 antibody (C-20, Santa Cruz Biotechnology), affinity-purified rabbit anti-GRP78 antibody (H-129, Santa Cruz Biotechnology), isotype-matched controls, or diluent only.

Techniques: Staining, Comparison, Immunohistochemical staining, Negative Control, Control

Journal: Cancer Research

Article Title: Phage Display–Derived Human Monoclonal Antibodies Isolated by Binding to the Surface of Live Primary Breast Cancer Cells Recognize GRP78

doi: 10.1158/0008-5472.can-06-4686

Figure Lengend Snippet: Figure 3. Biochemical characterization of human scFvs Ab39, Ab83, and Ab99. A, flow cytometry analysis of scFv phage antibodies Ab39 and Ab83 binding to different cancer cell lines. In one set of experiments, Ab39 and Ab83 were tested for binding to the four breast cancer cell lines MDA-MB-231, Hs578T, MDA-MB-157, and MCF-7. AbK2 was included as negative control. B, a second set of experiments assessed Ab39 binding to three other breast cancer cell lines, NM-2C5, M-4A4, and MDA-MB-157, and the Jurkat T-cell line. M13 phage was used as negative control. In both experiments, bound scFv phages were detected using a phycoerythrin- labeled anti-M13 antibody. C, inhibition of human monoclonal Ab39 staining by polyclonal anti-GRP78 antibody: Serial tissue sections of human melanoma (first column) and a ductal breast cancer (second column) were incubated with an anti-GRP78 antibody before adding Ab39. A marked reduction in Ab39 staining was observed with the C20 goat antibody directed against the COOH terminus of GRP78 (second row), whereas strong staining was observed when Ab39 was applied alone (first row), with a rabbit anti-GRP78 antibody, H-129 (third row), or with isotype-matched control antibody (fourth row). D, binding of scFv phage antibodies Ab39, Ab83, and Ab99 to bacterial expressed recombinant hamster GRP78 and bovine serum albumin (control) by ELISA.

Article Snippet: Tissue sections were processed and stained with Ab39, as described in the immunohistochemistry section above, except that before incubation with Ab39, sections were preincubated with affinity-purified goat anti-GRP78 antibody (C-20, Santa Cruz Biotechnology), affinity-purified rabbit anti-GRP78 antibody (H-129, Santa Cruz Biotechnology), isotype-matched controls, or diluent only.

Techniques: Flow Cytometry, Binding Assay, Negative Control, Labeling, Inhibition, Staining, Incubation, Control, Recombinant, Enzyme-linked Immunosorbent Assay

Journal: Clinical Science (London, England : 1979)

Article Title: IGFBP7 promotes endothelial cell repair in the recovery phase of acute lung injury

doi: 10.1042/CS20240179

Figure Lengend Snippet: ( A,B ) Detected by Western blotting, the protein expression of YAP1, P-YAP, TEAD1, and TEAD4 in HUVECs treated with Verteporfin (1 μM, a YAP1 inhibitor) for 48 h, simultaneously with a 24 h LPS treatment, internal control for normalization: β-Actin. ( C ) Assessed by qRT-PCR, the mRNA expression of CCNB1, CCNC, CCND1, CCNE1, SOX17, EGFR, and KI67 in HUVECs intervened with Verteporfin (1 μM) for 48 h, concurrently treated with LPS (5 μg/ml) for 24 h. ( D ) Determined by the CCK-8 assay, the cell viability of HUVECs intervened with Verteporfin (1 μM) for 48 h, concurrently treated with LPS (5 μg/ml) for 24 h. ( E,F ) Representative images of EDU-AF488 and DAPI co-staining in HUVECs intervened with Verteporfin (1 μM) for 48 h, concurrently treated with LPS (5 μg/ml) for 24 h. Scale bars: 50 μm (40×). ( G,H ) Detected by Western blotting, the protein expression of YAP1, P-YAP, TEAD1, and TEAD4 in HUVECs treated with rhIGFBP7 (1 μg/ml) for 48 h, simultaneously intervened with Verteporfin (1 μM) for 48 h, internal control for normalization: β-Actin. ( I ) Assessed by qRT-PCR, the mRNA expression of CCNB1, CCNC, CCND1, CCNE1, SOX17, EGFR, and KI67 in HUVECs treated with rhIGFBP7(1 μg/ml) for 48 h, simultaneously intervened with Verteporfin (1 μM) for 48 h. ( J ) Determined by the CCK-8 assay, the cell viability of HUVECs treated with rhIGFBP7 (1 μg/ml) for 48 h simultaneously intervened with Verteporfin (1 μM) for 48 h. ( K,L ) Representative images of EDU-AF488 and DAPI co-staining in HUVECs treated with rhIGFBP7 for 48 h, simultaneously intervened with Verteporfin (1 μM) for 48 h. Scale bars: 50 μm (40×). Data represented as means ± SDs. * P <0.05, ** P <0.01, *** P <0.005,**** P <0.001 [one-way ANOVA, Tukey’s test (B,C,D,F,H,I,J,L)]. CTRL, Control group. * represents the significance of Treat versus NC in the Control mouse model, and # represents the significance of Treat versus NC in the LPS-treated mouse model. #P<0.05".Please add " ##P<0.01.

Article Snippet: Verteporfin (YAP1 inhibitor) , MCE , Cat# HY-B0146.

Techniques: Western Blot, Expressing, Control, Quantitative RT-PCR, CCK-8 Assay, Staining

Journal: Clinical Science (London, England : 1979)

Article Title: IGFBP7 promotes endothelial cell repair in the recovery phase of acute lung injury

doi: 10.1042/CS20240179

Figure Lengend Snippet: ( A,B ) String and Genemania databases were used to predict the potential regulatory molecules of IGFBP7. ( C,D ) Detected by Western blotting, the protein expression of c-FOS in HUVECs intervened with rhIGFBP7 (0, 200, 500, 1000, and 2000 ng/ml) for 48 h, internal control for normalization: β-Actin. ( E,F ) Detected by Western blotting, the protein expression of c-FOS in HUVECs transfected with IGFBP7 siRNA for 48 h and treated with LPS (5 µg/ml) for 24 h, internal control for normalization: β-Actin. ( G,H ) Representative images of c-FOS-AF555 and DAPI co-staining in HUVECs transfected with IGFBP7 siRNA for 48 h. Scale bars: 50 μm (40×). ( I,J ) Representative images of c-FOS-AF555 and DAPI co-staining in HUVECs intervened with rhIGFBP7 (1 μg/ml) for 48 h. Scale bars: 50 μm (40×). ( K–L ) Detected by Western blotting, the protein expression of c-FOS in HUVECs treated with Verteporfin (1 μM, a YAP1 inhibitor) for 48 h, simultaneously with a 24 h LPS treatment, internal control for normalization: β-Actin. ( M,N ) Detected by Western blotting, the protein expression of c-FOS in HUVECs treated with rhIGFBP7 (1 μg/ml) for 48 h, simultaneously intervened with Verteporfin (1 μM) for 48 h, internal control for normalization: β-Actin. ( O,P ) Detected by Western blotting, the protein expression of C-FOS, YAP1, P-YAP, TEAD1, and TEAD4 in HUVECs treated with T5224 (40 μM, a C-FOS inhibitor) for 48 h, simultaneously with a 24 h LPS treatment, internal control for normalization: β-Actin. ( Q,R ) Detected by Western blotting, the protein expression of C-FOS, YAP1, P-YAP, TEAD1, and TEAD4 in HUVECs treated with rhIGFBP7 (1 μg/ml) for 48 h, simultaneously intervened with T5224 (40 μM, a C-FOS inhibitor) for 48 h, internal control for normalization: β-Actin. ( S,T ) Representative images of EDU-AF488 and DAPI co-staining in HUVECs intervened with T5224 (40 μM) for 48 h, concurrently treated with LPS (5 μg/ml) for 24 h. Scale bars: 50 μm (20×). ( U ) Assessed by qRT-PCR, the mRNA expression of CCNB1, CCNC, CCND1, CCNE1, SOX17, EGFR, and KI67 in HUVECs intervened with T5224 (40 μM) for 48 h, concurrently treated with LPS (5 μg/ml) for 24 h. ( V ) Determined by the CCK-8 assay, the cell viability of HUVECs intervened with T5224 (40 μM) for 48 h, concurrently treated with LPS (5 μg/ml) for 24 h. ( W,X ) Representative images of EDU-AF488 and DAPI co-staining in HUVECs treated with rhIGFBP7 (1 μg/ml) for 48 h and simultaneously intervened with T5224 (40 μM) for 48 h. Scale bars: 50 μm (40×). ( Y ) Assessed by qRT-PCR, the mRNA expression of CCNB1, CCNC, CCND1, CCNE1, SOX17, EGFR, and KI67 in HUVECs treated with rhIGFBP7 (1 μg/ml) for 48 h and simultaneously intervened with T5224 (40 μM) for 48 h. ( Z ) Determined by the CCK-8 assay, the cell viability of HUVECs treated with rhIGFBP7 (1 μg/ml) for 48 h and simultaneously intervened with T5224 (40 μM) for 48 h. Data represented as means ± SDs. * P <0.05, ** P <0.01, *** P <0.005,**** P <0.001 [one-way ANOVA, Tukey’s test (D,F,L,N,P,R,T,U,V,X,Y,Z)] and t -test (H,J). CTRL, Control group.

Article Snippet: Verteporfin (YAP1 inhibitor) , MCE , Cat# HY-B0146.

Techniques: Western Blot, Expressing, Control, Transfection, Staining, Quantitative RT-PCR, CCK-8 Assay

Journal: Clinical Science (London, England : 1979)

Article Title: IGFBP7 promotes endothelial cell repair in the recovery phase of acute lung injury

doi: 10.1042/CS20240179

Figure Lengend Snippet:

Article Snippet: Verteporfin (YAP1 inhibitor) , MCE , Cat# HY-B0146.

Techniques: Ubiquitin Proteomics, Recombinant, SYBR Green Assay, CCK-8 Assay, Extraction, Proliferation Assay, Immunoprecipitation

Journal: bioRxiv

Article Title: Immune-Epithelial Interactions via TGF-β Orchestrates Stem-Cell Niche Formation and Morphogenesis

doi: 10.1101/2025.05.22.655596

Figure Lengend Snippet: (a) Cytokine array profiling comparing co-cultures and epithelial-alone hydrogels shows elevated levels of IL-6, CXCL5, CCL2, TIMP-1, LIF, GRO-a (CXCL1), IL-8, IGFBP-1, IGFBP-2, VEGF-A, SDF-1a, and IGF-1in responder co-cultures with immune cells (n=2 patients). (b) Bar graph showing treatment of responder epithelial cells with individual (IL-6, CCL2, GRO-a (CXCL1), CXCL5, VEGF) or pooled recombinant cytokines at 2ng/ml. (n=8; ns, not significant, **p-value < 0.01, ***p-value < 0.001). (c) Bar graph showing treatment of responder epithelium with conditioned media from responder co-cultures (n=8, ns, not significant). (d) Flow cytometry analysis of patient single cells showing percent of CD45 immune cells out of all breast cells. (n=12; ns, not significant). (e) Bar graph and scatter plot showing percent of CD3 + CD4 - CD8 - double negative (DN) T cells out of all CD45 + immune cells (n=12; ***p-value < 0.001, two-tailed t-test). (f) Scatter plot showing percent of γδ T cells out of all DN T cells.

Article Snippet: For treatment with recombinant proteins IL-6 (PHC0066, Thermofisher,2ng/ml), CCL2 (279-MC-050/CF, R&D Systems, 2ng/ml), GRO-a (275-GR-010/CF, R&D Systems, 2ng/ml), CXCL5 (254-XB-025/CF, R&D Systems, 2ng/ml), VEGF (78073, Stem Cell Technologies, 2ng/ml), treatment began at timepoint 0 and continued for the entire culture period.

Techniques: Recombinant, Flow Cytometry, Two Tailed Test