Journal: Oxidative Medicine and Cellular Longevity

Article Title: Increased Gene Expression of RUNX2 and SOX9 in Mesenchymal Circulating Progenitors Is Associated with Autophagy during Physical Activity

doi: 10.1155/2019/8426259

Figure Lengend Snippet: Antibody list.

Article Snippet: Assessment of adipogenic stimuli was achieved by testing perilipin D1D8 (cat. #12589, Cell Signaling).

Techniques:

Journal: Investigative Ophthalmology & Visual Science

Article Title: Canonical and Truncated Transglutaminase-2 Drive TGF-β1 Autocrine Loop to Induce Fibrosis in Graves’ Orbitopathy

doi: 10.1167/iovs.66.5.22

Figure Lengend Snippet: Positive feedback loop between TGM2 and TGF-β1 drives fibrosis in OFs, and truncated TGM2 amplifies this effect. ( A ) Western blot analysis ( n control = 3, n test = 3) demonstrates the impact of various GTP concentrations on COL1A1 and α-SMA expression following TGF-β1 treatment (10 ng/mL). ( B ) Western blot results ( n control = 3, n test = 3) indicate that ZM39923 significantly affects the expression of COL1A1 and α-SMA induced by TGF-β1. ( C ) The expression changes of COL1A1 and α-SMA after TGF-β1 (10 ng/mL) treatment in the presence of A23187 are shown ( n control = 3, n test = 3). ( D ) FITC-tyramine staining reveals TGM2 activity ( n control = 3, n test = 3), while the right panel presents the quantitative results of TGM2 activity. S cale bar : 20 µm. ( E ) A colorimetric assay quantifies TGM2 activity following TGF-β1 treatment combined with various concentrations of ZM39923 and GTP ( n control = 3, n test = 3). ( F ) Colorimetric analysis evaluates the effects of ZM39923 and GTP on TGF-β1–induced TGM2 activity in cells where different TGM2 variants (TGM2_V1 and TGM2_V2) were reintroduced after TGM2 knockdown ( n control = 3, n test = 3). ( G ) Western blot analysis ( n control = 3, n test = 3) examines the effects of GTP or ZM39923 on COL1A1 and α-SMA expression after restoring full-length TGM2 (TGM2_V1) and truncated TGM2 variants (TGM2_V2) in fibroblasts with TGM2 knockdown. * P < 0.05, ** P < 0.01, *** P < 0.001 compared to the stimulation group; P < 0.05, P < 0.01, P < 0.001 compared to the control group (ANOVA), KD, knockdown; ns, not significant.

Article Snippet: Cells were treated with TGF-β1 (5 ng/mL; R&D Systems, Minneapolis, MN, USA); RepSox, ERKi, ZM39923, GTP, LDN-27219, cystamine, and A23187 (all from TargetMol, Boston, MA< USA); and rh-TGM2 (MedChemExpress, Monmouth Junction, NJ, USA) in DMEM with 1% FBS.

Techniques: Western Blot, Control, Expressing, Staining, Activity Assay, Colorimetric Assay, Knockdown

Journal: Investigative Ophthalmology & Visual Science

Article Title: Canonical and Truncated Transglutaminase-2 Drive TGF-β1 Autocrine Loop to Induce Fibrosis in Graves’ Orbitopathy

doi: 10.1167/iovs.66.5.22

Figure Lengend Snippet: The key role of the TGM2–TGF-β1 autocrine loop in fibrosis. ( A , B ) ELISA analysis ( n control = 3, n test = 3) shows that exogenous recombinant TGM2 (Rh-TGM2) significantly enhances the secretion of TGF-β1 in cell supernatants, exhibiting both time-dependent and dose-dependent effects. ( C ) TGM2 knockdown ( n = 3) markedly reduces TGF-β1 secretion, while TGM2 overexpression ( n = 3) significantly increases it. ( D , E ) Both GTP and ZM39923 treatments inhibit TGF-β1 secretion ( n control = 3, n test = 3). ( F , G ) Rescue experiments show that both ZM39923 and GTP suppress TGF-β1 autocrine secretion in cells expressing full-length TGM2 (TGM2_V1), while GTP does not affect TGF-β1 autocrine secretion in cells with the truncated form of TGM2 (TGM2_V2) ( n control = 3, n test = 3). ( H ) Western blotting results show that exogenous TGM2 activates the TGF-β1 signaling pathway via both SMAD-dependent and non-SMAD-dependent mechanisms ( n control = 3, n test = 3). ( I ) In OF cells with TGM2 knockdown, exposure to TGF-β1 (10 ng/mL) for 48 hours significantly inhibits TGF-β1 pathway activation ( n control = 3, n test = 3). ( J ) The model diagram illustrates the positive feedback loop of TGM2–TGF-β1 autocrine signaling: TGF-β1 upregulates TGM2 expression, enhancing TGF-β1 autocrine secretion, which further activates the TGF-β1 signaling pathway, leading to myofibroblast conversion and extracellular matrix deposition ( n control = 3, n test = 3). * P < 0.05, ** P < 0.01, *** P < 0.001 compared to the control group (ANOVA). ns, not significant.

Article Snippet: Cells were treated with TGF-β1 (5 ng/mL; R&D Systems, Minneapolis, MN, USA); RepSox, ERKi, ZM39923, GTP, LDN-27219, cystamine, and A23187 (all from TargetMol, Boston, MA< USA); and rh-TGM2 (MedChemExpress, Monmouth Junction, NJ, USA) in DMEM with 1% FBS.

Techniques: Enzyme-linked Immunosorbent Assay, Control, Recombinant, Knockdown, Over Expression, Expressing, Western Blot, Activation Assay

Journal: Science translational medicine

Article Title: Blockade of the CD93 pathway normalizes tumor vasculature to facilitate drug delivery and immunotherapy

doi: 10.1126/scitranslmed.abc8922

Figure Lengend Snippet: (A and B) Subcutaneous KPC (A, n = 12) and B16 melanoma (B, n = 10) tumors in mice were treated with CD93 mAb (7C10) twice a week when tumors became palpable. Data are representative of at least three independent experiments. (C to E) KPC tumor tissue was obtained on day 8 after antibody treatment. Blood vessel numbers within tumors were quantified by CD31 staining (C). Images and comparisons of tissues for blood vessels (CD31) expressing NG2 (D) and αSMA (E) to assess blood vessels covered by pericytes and smooth muscle cells, respectively. Data are representative of two independent experiments. Scale bars, 50 μm. DAPI, 4′,6-diamidino-2-phenylindole. (F to I) RNA-seq was performed on ECs isolated from KPC tumors in mice. Volcano plot revealed differentially expressed genes between control- and anti-CD93–treated groups (F). Gene ontology analysis was performed to identify biological pathways for differentially expressed genes (G). Quantitative PCR was performed to validate the expression increases of ALDH1A3, BNIP3, MAFA, and HMGCS2 (H), and the decreases of GATA1 and SFRP2 (I). (J and K) KPC tumor-bearing mice after 1 week of antibody treatment were assessed for lectin perfusion (J) and for dextran leakage (K). Data are representative of two independent experiments. Scale bars, 50 μm. (L) WT B6 mice were reconstituted with bone marrow cells from WT or CD93−/− mice. Eight weeks after reconstitution, mice were inoculated with B16 tumor cells and started with antibody treatment. Tumor volumes were measured at the time points shown after first treatment. n = 10 to 13. Data are representative of two independent experiments. Data are presented as means ± SEM. Data in (A), (B), and (L) were analyzed with two-way ANOVA test. Data in (C) to (F) and (H) to (K) were analyzed with unpaired Student’s t test. *P < 0.05, **P < 0.01, and ***P < 0.001. ns, not significant.

Article Snippet: Human IGFBP7 mAb (R003) and human CD93 mAb (MM01), obtained from Sino Biological, were used to block human IGFBP7-CD93 interaction.

Techniques: Staining, Expressing, RNA Sequencing Assay, Isolation, Real-time Polymerase Chain Reaction

Journal: Science translational medicine

Article Title: Blockade of the CD93 pathway normalizes tumor vasculature to facilitate drug delivery and immunotherapy

doi: 10.1126/scitranslmed.abc8922

Figure Lengend Snippet: (A) Graphic views of individual wells with a positive hit (IGFBP7) for CD93-Ig in a GSRA screening system. Scale bars, 50 μm. (B) HEK293T cells transfected with control (red) or CD93 construct (blue) were stained with IGFBP7-Ig for binding, with the presence of anti-CD93 (MM01) or anti-IGFBP7 (R003) mAb. Data are representative of two independent experiments. (C) HUVEC cells were stained with control (red) or IGFBP7-Ig (blue), with or without the presence of CD93 mAb (MM01). Data are representative of two independent experiments. (D) Microscale thermophoresis binding curve of human IGFBP7 to CD93 protein. (E) HEK293T cells transfected with control (red) or mouse CD93 construct (blue) were stained with mouse IGFBP7-Ig for binding, with the presence of anti-mouse CD93 (clone 7C10) or anti–mouse IGFBP7 (clone 2C6). Data are representative of at least three independent experiments. (F) CD93+ CHO cells were stained with MMRN2-Ig, with or without the presence of IGFBP7-His protein. Data are representative of two independent experiments. MFI, mean fluorescent intensity. (G) CD93+ CHO cells were stained for IGFBP7 binding, with or without the addition of MMRN2 protein. Data are representative of two independent experiments. (H) Wells coated with IGFBP7-His protein were incubated with CD93-His protein before examining for MMRN2-Ig binding by ELISA. Wells coated with CD93-His protein served as a positive control. Data are representative of two independent experiments. (I) Wells coated with mIGFBP7-His or anti-mCD93 (7C10) were incubated with mFc-tagged mCD93 fragment fusion proteins for binding by ELISA. Data are representative of two independent experiments. (J) HEK293T cells transfected with control (red) or CD93 construct (blue) were stained with MMRN2-Ig, with or without the presence of anti-mCD93 (7C10). Data are representative of two independent experiments. (K) Schematic diagrams represent the structure of a series of chimeric proteins that were generated by replacing each domain of IGFBP7 (BP7) with a corresponding portion from IGFBPL1 (BPL1). The binding of each chimeric protein to CD93 transfectant was tested by flow cytometry. Binding index refers to the ratio of mean fluorescence intensity of CD93 transfectant to control cells.

Article Snippet: Human IGFBP7 mAb (R003) and human CD93 mAb (MM01), obtained from Sino Biological, were used to block human IGFBP7-CD93 interaction.

Techniques: Transfection, Construct, Staining, Binding Assay, Microscale Thermophoresis, Incubation, Enzyme-linked Immunosorbent Assay, Positive Control, Generated, Flow Cytometry, Fluorescence

Journal: Science translational medicine

Article Title: Blockade of the CD93 pathway normalizes tumor vasculature to facilitate drug delivery and immunotherapy

doi: 10.1126/scitranslmed.abc8922

Figure Lengend Snippet: (A and B) Representative images of PD-L1 expression (green) in subcutaneous KPC tumors harvested on day 15 after antibody treatment. Scale bars, 50 μm. Single-cell suspensions prepared from KPC tumor tissues were quantified for PD-L1 expression by flow cytometry. (C to E) B6 mice with palpable KPC tumors were divided into four groups and treated with control, anti-CD93, anti-PD1, or the combination of anti-CD93 and anti-PD1 at the time points indicated by the red arrowheads. Tumor growth curve (C) and tumor weight 12 days after treatment (D) were measured. n = 10. The densities of respective immune cells within tumors were determined by flow cytometry (E). Data are representative of two independent experiments. (F to H) Mice with palpable B16 tumors were treated with control, anti-CD93, ICB (PD1 + CTLA mAbs), or the combination of anti-CD93 and ICB at the time points indicated by the red arrows. Tumor growth (F) and survival curve (G) of groups were shown. n = 10. The densities of CD45+ leukocytes and CD3+ T cells within tumors harvested on day 12 were determined by flow cytometry (H). Data are representative of two independent experiments. Data presented as means ± SEM. Data in (B) analyzed with unpaired Student’s t test. Data in (D), (E), and (H) analyzed with one-way ANOVA test with a Tukey post hoc test. Data in (C) and (F) analyzed with two-way ANOVA test with a Dunnett’s post hoc test. (G) Kaplan-Meier curves analyzed with log-rank (Mantel-Cox) test. *P < 0.05, **P < 0.01, and ***P < 0.001.

Article Snippet: Human IGFBP7 mAb (R003) and human CD93 mAb (MM01), obtained from Sino Biological, were used to block human IGFBP7-CD93 interaction.

Techniques: Expressing, Flow Cytometry