Journal: Cancer Science

Article Title: Characterization of myeloid signature genes for predicting prognosis and immune landscape in Ewing sarcoma

doi: 10.1111/cas.15688

Figure Lengend Snippet: The primer sequences of the candidate genes

Article Snippet: All tissue slices were subjected to a series of steps such as dewaxing, antigen retrieval, blocking, primary antibody incubation, and secondary antibody incubation, including CTSD (Proteintech, 21327‐1‐AP), SIRPA (Affinity, AF0253), FN1 (Proteintech, 15613‐1‐AP), CD8 (Proteintech, 66868‐1‐lg), CD206 (Proteintech, 60143‐1‐lg), CD66b (Abcam, ab300122), and CD57 (Servicebio, GB113461).

Techniques: Sequencing

Journal: Cancer Science

Article Title: Characterization of myeloid signature genes for predicting prognosis and immune landscape in Ewing sarcoma

doi: 10.1111/cas.15688

Figure Lengend Snippet: In vitro validation of CTSD, SIRPA, and FN1 expression. A, Western blotting showing protein expression levels of CTSD, SIRPA, and FN1 in Ewing sarcoma (EWS) cell lines and normal controls; B‐D, Quantitative real‐time PCR showing the relative mRNA expression levels of CTSD, SIRPA, and FN1 in EWS cell lines and normal controls. E‐G, Representative immunohistochemistry staining of CTSD, SIRPA, and FN1 in EWS tissue and matched para‐cancer. H‐J, Quantitative analysis of the positive rate of pathological stained sections for these three genes. * p < 0.05; ** p < 0.01; *** p < 0.001

Article Snippet: All tissue slices were subjected to a series of steps such as dewaxing, antigen retrieval, blocking, primary antibody incubation, and secondary antibody incubation, including CTSD (Proteintech, 21327‐1‐AP), SIRPA (Affinity, AF0253), FN1 (Proteintech, 15613‐1‐AP), CD8 (Proteintech, 66868‐1‐lg), CD206 (Proteintech, 60143‐1‐lg), CD66b (Abcam, ab300122), and CD57 (Servicebio, GB113461).

Techniques: In Vitro, Biomarker Discovery, Expressing, Western Blot, Real-time Polymerase Chain Reaction, Immunohistochemistry, Staining

Journal: Nature chemical biology

Article Title: An engineered Axl 'decoy receptor' effectively silences the Gas6-Axl signaling axis.

doi: 10.1038/nchembio.1636

Figure Lengend Snippet: Figure 1 | Engineering and characterization of receptor-based Axl antagonists. (a) Axl’s extracellular domain consists of two Ig-like domains containing high- and low-affinity Gas6 binding sites, followed by two fibronectin type III domains. Binding of Gas6 to Axl leads to receptor dimerization and activation of downstream signaling. Axl decoy receptors sequester Gas6, preventing activation of the Axl signaling cascade. (b) Overlaid flow cytometry dot plots representing binding of yeast-displayed wild-type Axl Ig1 (red) and unsorted Axl Ig1 library (blue) to 10 nM Gas6 (y axis) and expression levels on the yeast cell surface (x axis). (c) Flow cytometry histograms of the initial Axl library and intermediate sort products compared to wild-type Axl Ig1 (gray), measuring binding to 0.5 nM Gas6 (top row) and persistent Gas6 binding after a 30-h incubation with excess competitor (bottom row). MYD1 is also included for comparison. For clarity, only the gated population of yeast expressing Axl is shown. AU, arbitrary units. (d) Binding affinities of wild-type Axl Ig1, MYD1 and Axlnb to Gas6 as determined by KinExA. (e) Binding affinities to Gas6 of every permutation of the four mutations found in MYD1. Raw KinExA data and associated error values can be found in Supplementary Figures 2 and 3.

Article Snippet: After the appropriate incubation time, reactions were flowed over MYD1 Fc–coated beads, and captured free Gas6 was probed using a 500 ng/ml solution of antiHis6 Dylight 649 antibody (Rockland Immunochemicals Inc., 200-343-382).

Techniques: Binding Assay, Activation Assay, Flow Cytometry, Expressing, Incubation, Comparison

Journal: Nature chemical biology

Article Title: An engineered Axl 'decoy receptor' effectively silences the Gas6-Axl signaling axis.

doi: 10.1038/nchembio.1636

Figure Lengend Snippet: Figure 2 | Structural basis for high-affinity binding. (a) Gas6–MYD1 co-complex showing overall architecture and 2:2 stoichiometry. (b) MYD1 Ig1 (orange) and Gas6 LG1 (gray) domains showing the location of the four mutations in MYD1 with respect to the major binding site, which lies at the interface of these two domains. (c) Analysis of the wild-type structure (PDB code 2C5D) reveals steric crowding between the side chains of T457Gas6 and V92Axl. The V92A mutation alleviates this crowding in the MYD1 co-complex and facilitates local reorganization of side chains around V92A, exemplified by R48 and Q94. This in turn creates an elongated groove on MYD1 at the binding interface that allows reorientation of T457 on Gas6. (d) Reorientation of T457 results in capping of the N terminus of helix A. The wild-type (WT, green) and MYD1 (gray) structures are overlaid for comparison. (e) Capping stabilizes helix A, as seen by B-factor analysis (Online Methods).

Article Snippet: After the appropriate incubation time, reactions were flowed over MYD1 Fc–coated beads, and captured free Gas6 was probed using a 500 ng/ml solution of antiHis6 Dylight 649 antibody (Rockland Immunochemicals Inc., 200-343-382).

Techniques: Binding Assay, Mutagenesis, Comparison

Journal: Nature chemical biology

Article Title: An engineered Axl 'decoy receptor' effectively silences the Gas6-Axl signaling axis.

doi: 10.1038/nchembio.1636

Figure Lengend Snippet: Figure 4 | MYD1 Fc inhibits Axl activation and downstream signaling in skov3.ip cells. (a) Wild- type (WT) Axl Fc and MYD1 Fc, but not Axlnb Fc, can inhibit Gas6-mediated Axl activation in vitro. (b) Inhibition of Axl activation leads to reduced levels of phosphorylated Akt and Erk1/2 and an increase in the epithelial marker e-cadherin. For full (uncut) blots, see Supplementary Figure 11.

Article Snippet: After the appropriate incubation time, reactions were flowed over MYD1 Fc–coated beads, and captured free Gas6 was probed using a 500 ng/ml solution of antiHis6 Dylight 649 antibody (Rockland Immunochemicals Inc., 200-343-382).

Techniques: Activation Assay, In Vitro, Inhibition, Marker

Journal: Nature chemical biology

Article Title: An engineered Axl 'decoy receptor' effectively silences the Gas6-Axl signaling axis.

doi: 10.1038/nchembio.1636

Figure Lengend Snippet: Figure 5 | Sequestration of Gas6 by MYD1 Fc inhibits metastasis. (a) Amount of free Gas6 in serum of mice 12 h after administration of a single dose of MYD1 Fc. (b) Kinetics of Gas6 sequestration (black) and MYD1 Fc clearance (red) following a 1 mg per kg body weight dose of MYD1 Fc. (c) Using the off-rates of the Gas6-Axl Fc interactions (Fig. 3b), dissociation of Gas6 bound to either wild-type Axl Fc (red) or MYD1 Fc (blue) is plotted over time. The in vivo clearance of the Axl decoy receptors as measured in c is overlaid in black. Two mice were analyzed for each data point in b and c. (d–f) Tumor burden in in vivo models of metastatic human ovarian cancer. The number of visible metastases in animals treated with Axlnb Fc, wild-type Axl Fc or MYD1 Fc was counted in the skov3.ip (d) and OVCAR (f) tumor models. Representative images of mice from each treatment group in the skov3.ip model are shown, and arrows indicate disease (e). In both models, animals were administered 10 mg per kg body weight of the indicated protein twice weekly. (g) Lung metastases in the 4T1 luciferase breast cancer model, as quantified by ex vivo bioluminescent imaging. Mice received intravenous injections of the indicated treatment twice weekly. (h) Representative bioluminescent images of lungs and spleens from each treatment group; scale bar, 1 cm. Error bars represent ± s.d., n = 6–12 mice per group; *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Article Snippet: After the appropriate incubation time, reactions were flowed over MYD1 Fc–coated beads, and captured free Gas6 was probed using a 500 ng/ml solution of antiHis6 Dylight 649 antibody (Rockland Immunochemicals Inc., 200-343-382).

Techniques: In Vivo, Luciferase, Ex Vivo, Imaging