recombinant protein e1  (Native Antigen Inc)

Journal: Nature cell biology

Article Title: Crosstalk between small-cell lung cancer cells and astrocytes mimics brain development to promote brain metastasis

doi: 10.1038/s41556-023-01241-6

Figure Lengend Snippet: a. Volcano plot showing differentially expressed genes in hA co-cultured with NCI-H82 compared to hA alone. b. Gene ontology (GO) enrichment (top 9) for the genes that are upregulated in hA co-cultured with NCI-H82 compared to hA alone. c. Immunoassay (by WES capillary transfer) for SERPINE1 expression in naive hA and hA co-cultured with the NCI-H69 and SUBr1 human SCLC cell lines. HSP90 serves as a loading control. d. Quantification of (c) (n = 2 independent experiments). e. The expression level of SERPINE1 in astrocyte populations (as in Extended Data Fig. 2f) from N2N1G brain allograft tumor, tumor edge, and sham control brain. n = 3 biologically independent samples. f-g. Representative images and quantification of immunofluorescent staining of SERPINE1 in patient SCLC brain metastases. NCAM stains SCLC cells. GFAP stains astrocytes. DAPI stains nucleus. Scale bar, 20 μm. n = 3 samples. h. Ratio of active SERPINE1 measured by ELISA in culture medium from mouse astrocytes co-cultured with N2N1G cells for 2 days and then treated with DMSO (control) and Tiplaxtinin (5 μM). i. Cell viability (AlamarBlue assay) measured in N2N1G cells treated with recombinant mSERPINE1 and Tip (n = 3 experiments). j, k. Cell viability (AlamarBlue assay) measured in NCI-H69 cells ( j) and SUBr1 cells (k) treated with recombinant hSERPINE1 and the SERPINE1 inhibitor Tiplaxtinin (Tip) (n = 3 experiments). l-m. Relative cell viability (AlamarBlue assay) measured in NCI-H69 cells (l) and SUBr1 cells (m) cultured with hA compared to without hA, with or without Tiplaxtinin treatment (n = 3 experiments). n-o. Relative apoptosis (caspase3/7 activity) measured in NCI-H69 cells (n) and SUBr1 cells (o) cultured with hA compared to without hA, with or without Tip treatment (n = 3 experiments). p. Overlap between upregulated genes in NCI-H82 cultured with hA and N2N1G cells growing in the brain compared to subcutaneously. q. Apoptosis measured by cleaved caspase3/7 in shCtrl (control) and shReln N2N1G and 16T cells in culture. r. Immunoassay for SERPINE1 expression in N2N1G cells in Fig. 7m, n. Data show mean with SD. P values calculated via two-sided t-test when comparing two groups and one-way ANOVA when comparing three groups.

Article Snippet: Recombinant human Reelin 100 ng ml −1 (R&D Systems, 8546-MR-050) and mouse Reelin (R&D Systems, 3820-MR), human SERPINE1 (R&D Systems, 1786-PI) were used in these assays.

Techniques: Cell Culture, Expressing, Control, Staining, Enzyme-linked Immunosorbent Assay, Alamar Blue Assay, Recombinant, Activity Assay

Journal: Nature cell biology

Article Title: Crosstalk between small-cell lung cancer cells and astrocytes mimics brain development to promote brain metastasis

doi: 10.1038/s41556-023-01241-6

Figure Lengend Snippet: a, Quantification of tumour area after intracranial injection of N2N1G mouse cells with DMSO or tiplaxtinin (n = 6 tumours from 2 experiments) 14 days after injection. b,c, Representative images (b) and quantification (c) of immunofluorescence staining of GFAP in N2N1G allografts treated with DMSO control or tiplaxtinin 7 days after injection. n = 3 independent experiments. Scale bar, 20 μm. d,e, Representative immunofluorescence staining (d) and quantification of Casp3+ shCtrl and shReln N2N1G cells (e) growing in the brain of mice. n = 3 independent experiments. Scale bar, 20 μm. f,g, Representative immunofluorescence staining (f) and quantification (g) of Casp3+ shCtrl and shReln N2N1G cells overexpressing control vector or SERPINE1 (OE S1) when grown in the brain of mice. n = 3 independent experiments. Scale bar, 20 μm. h,i, Representative H&E images (h) and quantification (i) of tumour size from experiment in (f). Tumours appear dark purple. Scale bar, 2 mm. n = 3 independent experiments. j, Working model: SCLC is a neuroendocrine cancer whose neuronal features are often accentuated during tumour progression, including as SCLC cells reach the brain microenvironment. The neuronal programmes expressed by SCLC cells resemble those of neurons during early brain development and reactive astrocytes associated with SCLC cells in the brain also gain features of astrocytes during brain development. Secretion of the brain development molecule Reelin by SCLC cells recruits reactivated astrocytes to the brain metastasis site. Secretion of factors such as SERPINE1 by astrocytes in turn promotes the survival of SCLC cells.

Article Snippet: Recombinant human Reelin 100 ng ml −1 (R&D Systems, 8546-MR-050) and mouse Reelin (R&D Systems, 3820-MR), human SERPINE1 (R&D Systems, 1786-PI) were used in these assays.

Techniques: Injection, Immunofluorescence, Staining, Control, Plasmid Preparation

Journal: Nature cell biology

Article Title: Crosstalk between small-cell lung cancer cells and astrocytes mimics brain development to promote brain metastasis

doi: 10.1038/s41556-023-01241-6

Figure Lengend Snippet: a, Volcano plot showing differentially expressed genes in human astrocytes co-cultured with NCI-H69 cells compared to human astrocytes cultured alone. Selected genes are indicated. Padj values calculated by the Wald test with correction for multiple comparison. b, GO enrichment (top 9) for genes upregulated in human astrocytes co-cultured with NCI-H69 cells. c, Overlap between upregulated genes in tumour-associated mouse astrocytes and human astrocytes co-cultured with SCLC cells. The 11 overlapping genes are indicated. d, Representative immunofluorescence staining images of GFAP and SERPINE1 in a brain tumour from N2N1G mouse SCLC cells. Blue, DAPI DNA stain. Scale bar, 50 μm (top) and 10 μm (bottom). e, Quantification of SERPINE1 immunofluorescence intensity in astrocytes normalized to normal adult brain region from images as in (d) (N2N1G, n = 3 tumours). f,g, Relative viability (f) and apoptosis (g) of N2N1G cells cultured with mouse astrocytes with or without the SERPIN1 inhibitor tiplaxtinin (Tip, 5 μM) compared to N2N1G cells cultured alone. n = 3 independent experiments. h, Dose–response (measured cell viability via Alamar blue) of tiplaxtinin on mouse astrocytes. Black, naive astrocytes. Red, astrocytes reactivated by N2N1G cells. RLU, relative luminescence unit. Data are mean ± s.d. P values by two-sided t-test when comparing two groups and one-way ANOVA when comparing three or more groups; P value calculated by two-way ANOVA in h.

Article Snippet: Recombinant human Reelin 100 ng ml −1 (R&D Systems, 8546-MR-050) and mouse Reelin (R&D Systems, 3820-MR), human SERPINE1 (R&D Systems, 1786-PI) were used in these assays.

Techniques: Cell Culture, Comparison, Immunofluorescence, Staining