recombinant human gal1  (MedChemExpress)

Journal: Journal of Translational Medicine

Article Title: Cathepsin L -induced galectin-1 may act as a proangiogenic factor in the metastasis of high-grade serous carcinoma

doi: 10.1186/s12967-019-1963-7

Figure Lengend Snippet: Concentrations of treatments added

Article Snippet: Cells were starved, and subsequently treated ± recombinant human VEGF165 (20 ng/ml, positive control; Peprotech, London, UK), CathL (50 ng/ml; from human liver; Sigma-Aldrich, Poole, UK) and recombinant human Gal1 (50 ng/ml; Sigma-Aldrich, Poole, UK) ± NFκB, ERK1/2 and AKT inhibitors at their given concentrations (Table ) for 4, 10 min or 4 h. To confirm the effect of these inhibitors, HOMECs were pre-incubated with the inhibitors and then co-treated ± proangiogenic factors.

Techniques: Concentration Assay, Positive Control

Journal: Journal of Translational Medicine

Article Title: Cathepsin L -induced galectin-1 may act as a proangiogenic factor in the metastasis of high-grade serous carcinoma

doi: 10.1186/s12967-019-1963-7

Figure Lengend Snippet: CathL-induced Gal1 secretion in HOMECs is transcriptionally regulated via activation of NFκB. a Cells were seeded and starved overnight in media supplemented with 2% FCS. Cells were then treated ± CathL (50 ng/ml) and supernatants were collected after 4 min, 30 min, 2, 4, 8 and 24 h treatment. A commercially available ELISA kit was used to assess the levels of extracellular Gal1 using a SpectraMax plate reader. Results are mean ± SD and represented as fold change in secreted Gal1 vs control. n = 4–6. b Cells were treated ± CathL (50 ng/ml) and lysed after 6 or 24 h treatment. Real-time PCR was performed on extracted RNA using a Roche LightCycler 96 and the data were normalised to GAPDH and β2 M. c Gal1 secretion was assessed with or without CathL ± sulfasalazine (100 µmol/l) for 8 h and the supernates were analysed as above. d CathL induces activation of NFκB (p65) in HOMECs. Cells were incubated with sulfasalazine (sulf, 100 µmol/l) or media alone for 24 h and then treated ± CathL (50 ng/ml) or positive control TNF-α (160 pg/ml) and in the absence or presence of sulfasalazine (100 µmol/l). After 4 h treatment, the phosphorylated level of NFkB was assessed using a commercially available cell-based ELISA kit. Results are mean ± SD and are represented as fold change in phosho-NFkB relative to total NFkB (compared to control). e CathL induces an upregulation of LGALS1 gene expression via NFkB activation. After overnight incubation and pre-incubation, cells were treated with CathL ± sulfasalazine for 6 h and analysed for gene expression as above. Results are mean ± SD and are represented as fold change in LGALS1 gene expression (relative to control), n = 4. *p < 0.05, **p < 0.01 vs control. #p < 0.05, ##p < 0.01 vs CathL. n.s. denotes not significant

Article Snippet: Cells were starved, and subsequently treated ± recombinant human VEGF165 (20 ng/ml, positive control; Peprotech, London, UK), CathL (50 ng/ml; from human liver; Sigma-Aldrich, Poole, UK) and recombinant human Gal1 (50 ng/ml; Sigma-Aldrich, Poole, UK) ± NFκB, ERK1/2 and AKT inhibitors at their given concentrations (Table ) for 4, 10 min or 4 h. To confirm the effect of these inhibitors, HOMECs were pre-incubated with the inhibitors and then co-treated ± proangiogenic factors.

Techniques: Activation Assay, Enzyme-linked Immunosorbent Assay, Real-time Polymerase Chain Reaction, Incubation, Positive Control, In-Cell ELISA, Expressing

Journal: Journal of Translational Medicine

Article Title: Cathepsin L -induced galectin-1 may act as a proangiogenic factor in the metastasis of high-grade serous carcinoma

doi: 10.1186/s12967-019-1963-7

Figure Lengend Snippet: Gal1 induces HOMEC proliferation and activation of ERK1/2 and AKT. Cells were seeded in 2% gelatin pre-coated 96 well plates at a density of 10,000 cells/well in starvation media containing 2% FCS. a – c After overnight incubation, cells were treated ± Gal1 at various concentrations and incubated for 48 h. a WST-1 assay was used to assess cellular proliferation based on absorbance using a PHERAstar BMG plate-reader at 450 nm (n = 20). b Cell proliferation was examined at 50 ng/ml of Gal1 (CyQUANT). A commercially available CyQUANT reagent was used to assess cell proliferation after 48 h treatment based on fluorescence intensity using a FLUOstar BMG plate-reader at Ex/Em: 485/530 nm (n = 20). c A commercially available BrdU reagent was added to the wells for the last 24 h incubation and cellular proliferation was assessed (according to the manufacturer’s instructions) at 48 h based on absorbance using a SpectraMax plate-reader at 450/550 nm (n = 15). Results are mean ± SD and shown as percentage of the control, **p < 0.01 and ***p < 0.001 vs control (100%). After overnight incubation, cells were treated ± 50 ng/ml of Gal1 or 20 ng/ml of VEGF and incubated for 4 or 10 min. ERK1/2 ( d , e ) and AKT ( f , g ) phosphorylation was examined after 4 min ( d , f ) and 10 min ( e , g ) treatments. Commercially available cell-based ELISAs were used for the determination of ERK1/2 and AKT (S473) phosphorylation level. The ELISA experiments were carried out in quadruplets on two cell batches. The data show fold change in phosho-ERK1/2/AKT relative to total ERK1/2/AKT (compared to control). Results are mean ± SD, n.s., *p < 0.05, **p < 0.01 vs control (dotted lines); n = 4–6. n.s. denotes not significant vs control

Article Snippet: Cells were starved, and subsequently treated ± recombinant human VEGF165 (20 ng/ml, positive control; Peprotech, London, UK), CathL (50 ng/ml; from human liver; Sigma-Aldrich, Poole, UK) and recombinant human Gal1 (50 ng/ml; Sigma-Aldrich, Poole, UK) ± NFκB, ERK1/2 and AKT inhibitors at their given concentrations (Table ) for 4, 10 min or 4 h. To confirm the effect of these inhibitors, HOMECs were pre-incubated with the inhibitors and then co-treated ± proangiogenic factors.

Techniques: Activation Assay, Incubation, WST-1 Assay, CyQUANT Assay, Fluorescence, Enzyme-linked Immunosorbent Assay

Journal: Journal of Translational Medicine

Article Title: Cathepsin L -induced galectin-1 may act as a proangiogenic factor in the metastasis of high-grade serous carcinoma

doi: 10.1186/s12967-019-1963-7

Figure Lengend Snippet: Summary of the pro-proliferative effect of Gal1 on HOMECs at various concentrations (WST1 assay) (shown in Fig. 3 a)

Article Snippet: Cells were starved, and subsequently treated ± recombinant human VEGF165 (20 ng/ml, positive control; Peprotech, London, UK), CathL (50 ng/ml; from human liver; Sigma-Aldrich, Poole, UK) and recombinant human Gal1 (50 ng/ml; Sigma-Aldrich, Poole, UK) ± NFκB, ERK1/2 and AKT inhibitors at their given concentrations (Table ) for 4, 10 min or 4 h. To confirm the effect of these inhibitors, HOMECs were pre-incubated with the inhibitors and then co-treated ± proangiogenic factors.

Techniques: Concentration Assay

Journal: Journal of Translational Medicine

Article Title: Cathepsin L -induced galectin-1 may act as a proangiogenic factor in the metastasis of high-grade serous carcinoma

doi: 10.1186/s12967-019-1963-7

Figure Lengend Snippet: Gal1 induced HOMEC proliferation via the ERK1/2 and PI3K pathways, but not AKT pathway. Cells were seeded in 2% gelatin pre-coated 96 well plates at a density of 10,000 cells/well in starvation media containing 2% FCS. After overnight incubation, cells were treated ± Gal1 (50 ng/ml) and in the absence or presence of a U0126 (10 µmol/l) and PD98059 (25 µmol/L), b LY294002 (25 µmol/l) and MK2206 (5 µmol/l) and incubated for 72 h. Commercially available WST-1 assay was used to assess cellular proliferation. Results are mean ± SD and shown as percentage of the control, ***p < 0.001 vs control (100%), ###p < 0.001 vs Gal1 (normalised to control 100%), n = 14–15. n.s. denotes not significant

Article Snippet: Cells were starved, and subsequently treated ± recombinant human VEGF165 (20 ng/ml, positive control; Peprotech, London, UK), CathL (50 ng/ml; from human liver; Sigma-Aldrich, Poole, UK) and recombinant human Gal1 (50 ng/ml; Sigma-Aldrich, Poole, UK) ± NFκB, ERK1/2 and AKT inhibitors at their given concentrations (Table ) for 4, 10 min or 4 h. To confirm the effect of these inhibitors, HOMECs were pre-incubated with the inhibitors and then co-treated ± proangiogenic factors.

Techniques: Incubation, WST-1 Assay

Journal: Journal of Translational Medicine

Article Title: Cathepsin L -induced galectin-1 may act as a proangiogenic factor in the metastasis of high-grade serous carcinoma

doi: 10.1186/s12967-019-1963-7

Figure Lengend Snippet: Gal1 does not induce HOMEC migration via the ERK1/2 or PI3K/AKT pathway. HOMECs were seeded in the upper transwell chamber and treated ± Gal1 (50 ng/ml) in the absence or presence of ERK1/2 inhibitors a U0126 (10 µmol/l) and PD98059 (25 µmol/l), or PI3K and AKT inhibitors b LY294002 (25 µmol/l) and MK2206 (5 µmol/l) respectively in media containing 0.5% FCS. The lower well contained corresponding treatments. After 6 h, migrated cells were stained with calcein AM and fluorescence was quantified by using a FLUOstar plate reader at Ex/Em: 485/520. Results are mean ± SD and shown as percentage of the control, *p < 0.05, **p < 0.01, ***p < 0.001 vs control (100%), n = 4–11. n.s. denotes not significant

Article Snippet: Cells were starved, and subsequently treated ± recombinant human VEGF165 (20 ng/ml, positive control; Peprotech, London, UK), CathL (50 ng/ml; from human liver; Sigma-Aldrich, Poole, UK) and recombinant human Gal1 (50 ng/ml; Sigma-Aldrich, Poole, UK) ± NFκB, ERK1/2 and AKT inhibitors at their given concentrations (Table ) for 4, 10 min or 4 h. To confirm the effect of these inhibitors, HOMECs were pre-incubated with the inhibitors and then co-treated ± proangiogenic factors.

Techniques: Migration, Staining, Fluorescence

Journal: Journal of Translational Medicine

Article Title: Cathepsin L -induced galectin-1 may act as a proangiogenic factor in the metastasis of high-grade serous carcinoma

doi: 10.1186/s12967-019-1963-7

Figure Lengend Snippet: High positive correlation between Gal1 expression and number of vessels, and area of vessels. a Expression of Gal1 (upper panel) and CD34 (lower panel) in endothelium (moderate expression- red arrows) in control (left), SC wo MTS (serous carcinoma without omental metastasis, moderate expression; middle), and SC w MTS (serous carcinoma with omental metastasis, high expression; right). In SC w MTS, Gal1 expression is shown in endothelium (red arrows), cancer cells (orange arrows) and stromal lymphocytes infiltration (yellow arrows). b Significantly higher Gal1 expression in SC w MTS group compared to control and SC wo MTS. Gal1 expression is represented as an intensity score, as described by Chen et al. . c A significant increase in the number of vessels and d area of vessels were found in SC w MTS group compared to the other groups. The vessels were counted according to Weidner’s method described in “ ” section . e Very high correlation between Gal1 and number, and area of vessels, indicating a potential proangiogenic role for Gal1. f Univariate and g multivariate hazard ratios of Cox analysis of predictive patients’ survival. Photographs were taken using a Nikon Eclipse 50i. ***p < 0.001 vs control group, ###p < 0.001 vs SC wo MTS. n.s. denotes not significant. Magnification: ×200

Article Snippet: Cells were starved, and subsequently treated ± recombinant human VEGF165 (20 ng/ml, positive control; Peprotech, London, UK), CathL (50 ng/ml; from human liver; Sigma-Aldrich, Poole, UK) and recombinant human Gal1 (50 ng/ml; Sigma-Aldrich, Poole, UK) ± NFκB, ERK1/2 and AKT inhibitors at their given concentrations (Table ) for 4, 10 min or 4 h. To confirm the effect of these inhibitors, HOMECs were pre-incubated with the inhibitors and then co-treated ± proangiogenic factors.

Techniques: Expressing

Journal: Journal of Translational Medicine

Article Title: Cathepsin L -induced galectin-1 may act as a proangiogenic factor in the metastasis of high-grade serous carcinoma

doi: 10.1186/s12967-019-1963-7

Figure Lengend Snippet: A summary of the proposed CathL-induced Gal1 secretion pathway and its pro-proliferative function and potential therapeutic targets in HOMECS. 1. CathL non-proteolytically activates a cell surface receptor, possibly a receptor tyrosine kinase, which leads to an increase in activated levels of the transcription factor NFκB, resulting in increased expression of LGALS1 (Gal1 mRNA). Sulfasalazine, an inhibitor of NFκB, reduces LGALS1 expression and subsequent secreted level of Gal1. 2. LGALS1 is transcribed and translated into Gal1 which is secreted out of the cell, possibly utilising a Na + /K + ATP-pump, via a non-conventional secretory pathway. 3. Secreted Gal1 induces activation of MAP kinase ERK1/2 and PI3K/AKT via an autocrine mechanism and an unknown receptor(s), where the ERK1/2 pathway induces HOMEC proliferation. The MEK/ERK1/2 inhibitors U0126 and PD98059 significantly reduce this cellular function by inhibiting ERK1/2 phosphorylation. Both the PI3K inhibitor LY294002 and AKT inhibitor MK2206 inhibit phosphorylation of AKT at Ser473 (S473) in Gal1-treated HOMECs. However, only LY294002, but not MK2206, inhibits Gal1-induced HOMEC proliferation, suggesting a cross-reactivity of PI3K inhibitor LY294002 with the ERK1/2 pathway. Potential therapeutic targets are: Gal1 secretory mechanism, Gal1 binding to its receptor(s), and blocking receptor(s) for Gal1. N, nucleus; C, cytoplasm

Article Snippet: Cells were starved, and subsequently treated ± recombinant human VEGF165 (20 ng/ml, positive control; Peprotech, London, UK), CathL (50 ng/ml; from human liver; Sigma-Aldrich, Poole, UK) and recombinant human Gal1 (50 ng/ml; Sigma-Aldrich, Poole, UK) ± NFκB, ERK1/2 and AKT inhibitors at their given concentrations (Table ) for 4, 10 min or 4 h. To confirm the effect of these inhibitors, HOMECs were pre-incubated with the inhibitors and then co-treated ± proangiogenic factors.

Techniques: Cell Surface Receptor Assay, Expressing, Activation Assay, Cell Function Assay, Binding Assay, Blocking Assay