Journal: Journal of Molecular Endocrinology

Article Title: Sensitivity of human granulosa cell tumor cells to epidermal growth factor receptor inhibition

doi: 10.1530/jme-13-0286

Figure Lengend Snippet: Figure 4 EGFR inhibition reduces cell number and activates caspase-3/7 and apoptosis in ex vivo granulosa cell tumor cells. (A) Changes in cell viability in response to treatment with gefitinib were analyzed by WST-1 in primary GCT cells (nZ4). (B) Caspase-3/7 activity was measured in primary-cultured GCT cells after 48-h treatment with either 0.1% DMSO, 10 ng/ml EGF, or 10 mM gefitinib (nZ3). (C) KGN cells and primary GCT cells grown on glass chamber slides were treated with either DMSO or 3 mM (KGN) or 10 mM (GCT) doses of gefitinib and stained with DAPI. The number of apoptotic cells was counted based on nuclear morphology and is expressed as a percentage of total cell number. (D) The figure shows DMSO- and gefitinib- treated primary-cultured GCTcells from a representative experiment, white arrows indicate apoptotic cells. Data represent the meanGS.E.M. of three independent experiments (A and B). *P!0.05; **P!0.01; Student’s t-test.

Article Snippet: Nonspecific binding was blocked with 5% nonfat milk or Published by Bioscientifica Ltd. Jo u rn a l o f M o le cu la r E n d o cr in o lo g y 3% BSA in Tris-buffered saline with 0.1% Tween for 1 h. The following antibodies were used: rabbit a-EGFR 1:5000 (100-401-149; Rockland Immunochemicals, PA, USA), rabbit a-phospho-EGFR 1:10 000 (ab40815, Abcam), rabbit a-phospho-AKT 1:1000, and rabbit a-AKT 1:1000 (#9271 and #4691 respectively, Cell Signaling Technology, Inc., Danvers, MA, USA) rabbit a-ACTIVE-MAPK (detecting ERK1/2) proteins 1:5000 (V803A, Promega Corporation), rabbit p44/42 MAPK (ERK1/2) 1:1000 (#9102, Cell Signaling Technology, Inc.), and goat a-actin IgG 1:5000 (sc-1616, Santa Cruz Biotechnology, Inc.).

Techniques: Inhibition, Ex Vivo, Activity Assay, Cell Culture, Staining

Journal: eLife

Article Title: High-phytate/low-calcium diet is a risk factor for crystal nephropathies, renal phosphate wasting, and bone loss

doi: 10.7554/elife.52709

Figure Lengend Snippet: Figure 6. Phytate-mediated Ca2+ deficiency promotes vitamin D insufficiency and renal phosphate wasting independent of FGF23 expression. (A–C) Time-course analysis of serum levels of intact PTH (A), 25(OH)D (B), and 1,25(OH)2D (C) in rats fed control, HP-LCa2+, and HP-HCa2+ diets. For the early measurement of 25(OH)D (B) and 1,25(OH)2D, we pooled the sera from 2 to 3 rats. (D–F) Time-course analysis of renal CYP27B1 (D), CYP24A1 (E), and VDR (F) in rats fed control, HP-LCa2+, and HP-HCa2+ diets. (G) Immunoblot analysis of renal aKlotho, NHERF1, NaPi-2a in rats fed control, HP-LCa2+, Figure 6 continued on next page

Article Snippet: Key resources table Reagent type (species) or resource Designation Source or reference Identifiers Additional information Genetic reagent (Rattus norvegicus) Sprague Dawley rat Orient Bio, Seoul, Korea Developed by Sprague Dawley, Inc. Chemical compound, drug AIN-93G Dyets Inc. DYET# 10700 Control diet Chemical compound, drug Phytic acid Sigma-Aldrich Cat. #: P-8810 Phytate diet Chemical compound, drug HNO3 Sigma-Aldrich Cat. #: 438073 Fecal mineral analysis Chemical compound, drug HF Sigma-Aldrich Cat. #: 695068 Fecal mineral analysis Chemical compound, drug HCl Sigma-Aldrich Cat. #: H1758 Fecal mineral analysis Chemical compound, drug EDTA Sigma-Aldrich Cat. #: 93283 Fecal phytate analysis Chemical compound, drug NaOH Sigma-Aldrich Cat. #: 415413 Fecal phytate analysis Chemical compound, drug Calcium Beckman Coulter OSR6113 Serum/urine biochemistry Chemical compound, drug Phosphate Beckman Coulter OSR6122 Serum/urine biochemistry Chemical compound, drug BUN (Urea) Beckman Coulter OSR6134 Serum/urine biochemistry Chemical compound, drug Creatinine Beckman Coulter OSR6178 Serum/urine biochemistry Chemical compound, drug Magnesium Beckman Coulter OSR6189 Serum/urine biochemistry Chemical compound, drug Urine protein Beckman Coulter OSR6170 Urine biochemistry Commercial assay or kit Rat intact PTH Immutopics Cat. #: 60–2500 ELISA kit Commercial assay or kit Human PTH Abcam Cat. #: ab230931 ELISA kit Commercial assay or kit Rat soluble RANKL Immundiagnostik Cat. #: K1019 ELISA kit Commercial assay or kit Rat osteoprotegerin Alpco Immunoassay Cat. #: 30–1020 ELISA kit Commercial assay or kit Rat intact FGF23 Elabscience Cat. #: E-EL-R3031 ELISA kit Commercial assay or kit Rat C-terminal FGF23 Elabscience Cat. #: E-EL-RB0377 ELISA kit Commercial assay or kit Human FGF23 Elabscience Cat. #: E-EL-H1116 ELISA kit Commercial assay or kit 25(OH)-Vitamin D Abbott/USA ARCHITECT 25-OH Vitamin D Chemiluminescence microparticle immunoassay Commercial assay or kit 1,25-(OH)two vitamin D DIAsoure 1,25(OH)2-VIT, D-RIA-CT/Belgium Radioimmunoassay Continued on next page Kim et al. eLife 2020;9:e52709.

Techniques: Expressing, Control, Western Blot

Journal: Molecules

Article Title: Revealing Novel Source of Breast Cancer Inhibitors from Seagrass Enhalus acoroides : In Silico and In Vitro Studies

doi: 10.3390/molecules29051082

Figure Lengend Snippet: ΔG of Molecular docking parameter of identified compounds of Seagrass E. acoroides .

Article Snippet: In vitro analysis of HIF-1α, EGFR tyrosine kinase, and HER2 Expressions was carried out in accordance with the manufacturer’s protocol (HIF-1 alpha Monoclonal Antibody (ESEE122), eBioscienceTM; Human EGFR (Epidermal Growth Factor Receptor) ELISA Kit; Elabscience ® for HER2) and established research experimental guidelines [ ].

Techniques: Control

Journal: Molecules

Article Title: Revealing Novel Source of Breast Cancer Inhibitors from Seagrass Enhalus acoroides : In Silico and In Vitro Studies

doi: 10.3390/molecules29051082

Figure Lengend Snippet: Downregulation of HIF-1A, EGFR tyrosine kinase, and HER2 by Seagrass EAE.

Article Snippet: In vitro analysis of HIF-1α, EGFR tyrosine kinase, and HER2 Expressions was carried out in accordance with the manufacturer’s protocol (HIF-1 alpha Monoclonal Antibody (ESEE122), eBioscienceTM; Human EGFR (Epidermal Growth Factor Receptor) ELISA Kit; Elabscience ® for HER2) and established research experimental guidelines [ ].

Techniques:

Journal: Bone research

Article Title: Metformin accelerates bone fracture healing by promoting type H vessel formation through inhibition of YAP1/TAZ expression.

doi: 10.1038/s41413-023-00279-4

Figure Lengend Snippet: Fig. 1 Metformin promotes angiogenesis under hypoxic conditions in vitro. Representative images (a) of the transwell migration assay with quantification of crystal violet-stained migrated HMECs (b) treated with metformin under different oxygen concentrations (21% and 1% O2). Met: metformin. Scale bar: 100 μm. n = 3 per group. Representative images (c) of tube formation of HMECs on Matrigel with quantification of the total branching points (d), total tube length (e), and total loop numbers (f). Scale bar: 200 μm. n = 3 per group. g CCK-8 analysis of the proliferation of HMECs. n = 4 per group. h qRT‒PCR analysis of the mRNA levels of the HIF-1α target genes Vegfa and Lrg1 in HMECs with or without metformin treatment under different oxygen concentrations. n = 3 per group. Data are presented as the mean ± SEM. *P < 0.05, **P < 0.01, and ***P < 0.001

Article Snippet: Enzyme-linked immunosorbent assay (ELISA) The concentrations of OCN and VEGFA were determined using the Mouse OCN (Elabscience, E-EL-M0864c, Wuhan, China) or VEGFA (Elabscience, E-EL-M1292c) ELISA kit according to the manufacturers’ instructions.

Techniques: In Vitro, Transwell Migration Assay, Staining, CCK-8 Assay

Journal: Bone research

Article Title: Metformin accelerates bone fracture healing by promoting type H vessel formation through inhibition of YAP1/TAZ expression.

doi: 10.1038/s41413-023-00279-4

Figure Lengend Snippet: Fig. 3 Metformin promotes type H vessel formation in osteoporotic fracture mice. a Representative CD31 and Emcn coimmunostaining images (left) with quantification of the type H vessel ratio in calluses (right) from the osteoporotic mice treated with PBS, Met, PTH, and ALN at 3, 6, and 9 weeks post-fracture. ca: callus. The dotted line represents the boundary of the callus. Met metformin, ALN alendronate, PTH parathyroid hormone. Scale bar: 100 μm. n = 5 per group. b Representative Ki67 and Emcn coimmunostaining images (left) with quantification of the number of Ki67-positive endothelial cells in calluses (right) from the osteoporotic mice treated with PBS, Met, PTH, and ALN at 3, 6, and 9 weeks post-fracture. Scale bar: 100 μm. n = 5 per group. ELISAs for the serum (c) and bone marrow (d) concentrations of VEGFA at 9 weeks post-osteoporotic fracture. n = 8 per group; Data are presented as the mean ± SEM. *P < 0.05, **P < 0.01, and ***P < 0.001

Article Snippet: Enzyme-linked immunosorbent assay (ELISA) The concentrations of OCN and VEGFA were determined using the Mouse OCN (Elabscience, E-EL-M0864c, Wuhan, China) or VEGFA (Elabscience, E-EL-M1292c) ELISA kit according to the manufacturers’ instructions.

Techniques:

Journal: Bone research

Article Title: Metformin accelerates bone fracture healing by promoting type H vessel formation through inhibition of YAP1/TAZ expression.

doi: 10.1038/s41413-023-00279-4

Figure Lengend Snippet: Fig. 4 Metformin promotes the expression of HIF-1α by inhibiting the expression of YAP1/TAZ in HMECs under hypoxic conditions. a qRT‒PCR analysis of the inhibitory efficiency of siRNAs targeting HIF-1α. n = 3 per group. b qRT‒PCR analysis of the expression of HIF-1α and its target genes Vegfa and Lrg1 in the si-HIF-1α-transfected HMECs with or without metformin treatment under hypoxic conditions (1% O2). Met: metformin. n = 3 per group. Immunofluorescence staining images and quantification showing the protein levels of HIF-1α (c), YAP1 (d), and TAZ (e) in the HMECs treated with PBS (control) or metformin under hypoxic conditions. Scale bar: 20 μm. n = 9 per group. f qRT‒PCR analysis of the inhibitory efficiency of siRNAs targeting YAP1 or TAZ. n = 3 per group. g Immunofluorescence staining images and quantification showing the protein level of HIF-1α in the hypoxia-cultured HMECs from the si-Con, si-YAP1, si-TAZ, si-Y/T, and si-Y/T + Met groups. Y/T: YAP1 and TAZ. Scale bar: 20 μm. n = 3 per group. Data are presented as the mean ± SEM. *P < 0.05, **P < 0.01, and ***P < 0.001

Article Snippet: Enzyme-linked immunosorbent assay (ELISA) The concentrations of OCN and VEGFA were determined using the Mouse OCN (Elabscience, E-EL-M0864c, Wuhan, China) or VEGFA (Elabscience, E-EL-M1292c) ELISA kit according to the manufacturers’ instructions.

Techniques: Expressing, Transfection, Staining, Control, Cell Culture

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