Journal: Chinese Journal of Cancer Research

Article Title: Tim-3 promotes cell aggressiveness and paclitaxel resistance through NF-κB/STAT3 signalling pathway in breast cancer cells

doi: 10.21147/j.issn.1000-9604.2020.05.02

Figure Lengend Snippet: Effect of Tim-3 OE on tube formation of endothelial cells. (A) Tube formation ability of HUVECs cultured in conditioned medium from MDA-MB-231 Tim-3-overexpressing cells (P=0.014 vs . Scr); (B) Tube formation ability of HUVECs cultured in conditioned medium from MCF7 Tim-3-overexpressing cells (P=0.016 vs . Scr); (C) Protein levels of VEGFA, VEGFB and VEGFD following Tim-3 OE (left) and quantitative densitometric analysis (right); (D) mRNA expression of VEGFA and VEGFD genes in breast cancer cells. OE, overexpression; Tim-3, T-cell immunoglobulin and mucin-domain containing molecule-3; HUVEC, human umbilical vein endothelial cell; VEGF, vascular endothelial growth factor. * , P<0.05; ** , P<0.01; *** , P<0.001.

Article Snippet: ELISA was performed using the human VEGFC (cat. no. E-EL-H1600; Elabscience) and vascular endothelial growth factor receptor 2 (VEGFR2) ELISA kit (cat. no. E-EL-H1603; Elabscience), according to the manufacturer’s protocol.

Techniques: Cell Culture, Expressing, Over Expression

Journal: Chinese Journal of Cancer Research

Article Title: Tim-3 promotes cell aggressiveness and paclitaxel resistance through NF-κB/STAT3 signalling pathway in breast cancer cells

doi: 10.21147/j.issn.1000-9604.2020.05.02

Figure Lengend Snippet: VEGFC and VEGFR2 protein levels in conditioned media of stable cell lines indicated by ELISA. (A) VEGFC; (B) VEGFR2. VEGF, vascular endothelial growth factor; ELISA, enzyme-linked immunosorbent assay.

Article Snippet: ELISA was performed using the human VEGFC (cat. no. E-EL-H1600; Elabscience) and vascular endothelial growth factor receptor 2 (VEGFR2) ELISA kit (cat. no. E-EL-H1603; Elabscience), according to the manufacturer’s protocol.

Techniques: Stable Transfection, Enzyme-linked Immunosorbent Assay

Journal: Chinese Journal of Cancer Research

Article Title: Tim-3 promotes cell aggressiveness and paclitaxel resistance through NF-κB/STAT3 signalling pathway in breast cancer cells

doi: 10.21147/j.issn.1000-9604.2020.05.02

Figure Lengend Snippet: Schematic illustration of role of Tim-3 in breast cancer. Upregulation of Tim-3 not only promotes cell proliferation, migration and invasion, but also disrupts cell-cell tight junction, increases angiogenesis of endothelial cells and paclitaxel-resistance. Tim-3 functions in breast cancer cells by activating NF-κB/STAT3 pathway and downstream target genes. Tim-3, T-cell immunoglobulin and mucin-domain containing molecule-3; IL-6, interleukin 6; ZO, zona occludens; VEGF, vascular endothelial growth factor; CCND1, cyclin D1; MMP-1, matrix metalloproteinase-1.

Article Snippet: ELISA was performed using the human VEGFC (cat. no. E-EL-H1600; Elabscience) and vascular endothelial growth factor receptor 2 (VEGFR2) ELISA kit (cat. no. E-EL-H1603; Elabscience), according to the manufacturer’s protocol.

Techniques: Migration

Journal: PLoS ONE

Article Title: N-Terminal Cleavage and Release of the Ectodomain of Flt1 Is Mediated via ADAM10 and ADAM 17 and Regulated by VEGFR2 and the Flt1 Intracellular Domain

doi: 10.1371/journal.pone.0112794

Figure Lengend Snippet: Panel A: HEK293 cells transfected with VEGFR2 and conditioned media immunoblotted for sFlt1. VEGFR2 expression dose dependently increases sFlt1 expression. Panel B: HEK293 cells transfected with VEGFR2 and VEGF in conditioned media measured by ELISA. VEGFR2 dose dependently reduces free VEGF in conditioned media. **p<0.001 against vehicle, $ p<0.05 between low and high VEGFR2 expressed groups, n = 4. Panel C: HEK293 cells co-transfected with epitope tagged Flt1 ΔCTD and VEGFR2 and Flt1 ΔCTD (Flag epitope) and its N-term fragment (HA epitope) measured in cell lysates and conditioned media respectively. Cell lysates were also immunoblotted for VEGFR2 to confirm overexpression and for tubulin as a control for loading. VEGFR2 dose dependently reduces cleavage of Flt1 manifest by increased abundance of uncleaved Flt1 in cell lysates and reduced abundance of the cleaved N-terminal fragment in conditioned media (condt. Media). Panel D: HEK293 cells co-transfected with epitope tagged Flt1 ΔCTD and VEGFR2 or control plasmid and treated with 30 nM PMA in the presence and absence of 10 U/ml heparin overnight. Conditioned media (Condt. Media) was immunoblotted for the N-terminal fragment while cell lysates were immunoprecipitated with Flag and then blotted for VEGFR2 (Flk1). VEGFR2 significantly reduces the availability of the cleaved Flt1 fragment in conditioned media while heparin has a modest effect. VEGFR2 and Flt1 physically associate and heparin does not alter this association.

Article Snippet: C-terminal GFP-tagged open reading frame (ORF) clone of full length human VEGFR2 was purchased from OriGene (Rockville, MD).

Techniques: Transfection, Expressing, Enzyme-linked Immunosorbent Assay, FLAG-tag, Over Expression, Control, Plasmid Preparation, Immunoprecipitation