Journal: Oncotarget

Article Title: The role of prospero homeobox 1 (PROX1) expression in follicular thyroid carcinoma cells

doi: 10.18632/oncotarget.23167

Figure Lengend Snippet: NTHY: normal thyroid, BcPAP and TPC1: papillary thyroid carcinoma-derived cell lines, FTC-133 and CGTH-W-1: follicular thyroid carcinoma-derived cell lines. (A) Relative PROX1 mRNA expression in all cell lines. PROX1 and β -actin mRNA levels were quantified and PROX1 expression normalized against the expression of the ACTB housekeeping gene. Each bar represents the mean of triplicate measurements on three different samples for each cell line. Statistical significance was evaluated by paired Student’s t-test using the GraphPad Prism software. The corresponding P -value ( **** : P -value < 0.0001) is presented. (B) The Prox1 protein levels in the tested cell lines by Western blot. Total protein extracts from logarithmically growing cells were analyzed. ß-actin was used as a loading control. (C) Immunofluorescent labeling of the Prox1 protein (red) and its cellular localization in the analyzed cell lines. Inset: the Prox1 content and cellular localization after DAPI (blue) removal (scale bar: 10μm). All pictures were taken at a x 1000 magnification. Below: Prox1 protein content in nuclear (n) and cytoplasmic (c) fractions determined by Western blot. (D) Immunoblot analysis of Hsp90 (control for the cytoplasmic fraction) and PARP1 (control for nuclear fraction) protein expression in nuclear (n) and cytoplasmic (c) fractions isolated from the HepG2 cells.

Article Snippet: Prox1 , Goat anti-human Prox1 (1:2000), R&D System, USA , Affinity-purified rabbit anti-goat antibody/HRP (1:20000), Jackson ImmunoResearch Laboratories, USA.

Techniques: Derivative Assay, Expressing, Software, Western Blot, Control, Labeling, Isolation

Journal: Oncotarget

Article Title: The role of prospero homeobox 1 (PROX1) expression in follicular thyroid carcinoma cells

doi: 10.18632/oncotarget.23167

Figure Lengend Snippet: FTC-133 cells were treated with LY204002 (A) , the PI3K inhibitor (50μM), or with A6730 (B) , the AKT1/AKT2 inhibitor (10 μM), for 48 hours. Negative control cells were grown with DMSO instead of the inhibitors. (C) AKT and phospho-AKT expression in FTC-133 cells after silencing of PROX1 . The whole cell protein extracts were prepared and analyzed by Western blotting using a primary antibody against phospho-AKT (Ser473), AKT, or Prox1, followed by an incubation with relevant goat anti-rabbit or goat-anti mouse HRP-conjugated affinity-purified secondary antibodies. β-actin was used as a loading control.

Article Snippet: Prox1 , Goat anti-human Prox1 (1:2000), R&D System, USA , Affinity-purified rabbit anti-goat antibody/HRP (1:20000), Jackson ImmunoResearch Laboratories, USA.

Techniques: Negative Control, Expressing, Western Blot, Incubation, Affinity Purification, Control

Journal: Oncotarget

Article Title: The role of prospero homeobox 1 (PROX1) expression in follicular thyroid carcinoma cells

doi: 10.18632/oncotarget.23167

Figure Lengend Snippet: PROX1 expression in FTC-133 cells following PROX1 silencing with PROX1 -specific siRNA (A–C) or PROX1 overexpression (D–F). siNEG: negative control (cells transfected with negative siRNA). (A) and (D) PROX1 mRNA levels determined by real-time quantitative PCR (RT-qPCR). The results were normalized against ACTB transcript levels and bars represent the average PROX1 transcript levels in cells transfected with PROX1 -siRNA compared to control cells. The results represent means from five independent experiments. (B) and (E) Prox1 protein levels determined by Western blotting, and (C and F) by immunofluorescent staining. Prox1 was detected with a rabbit anti-Prox1 polyclonal antibody and the DyLight 594-conjugated purified rabbit F(ab) 2 anti-goat secondary antibodies (red), and the nuclei were stained with DAPI (blue).

Article Snippet: Prox1 , Goat anti-human Prox1 (1:2000), R&D System, USA , Affinity-purified rabbit anti-goat antibody/HRP (1:20000), Jackson ImmunoResearch Laboratories, USA.

Techniques: Expressing, Over Expression, Negative Control, Transfection, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Control, Western Blot, Staining, Purification

Journal: Oncotarget

Article Title: The role of prospero homeobox 1 (PROX1) expression in follicular thyroid carcinoma cells

doi: 10.18632/oncotarget.23167

Figure Lengend Snippet: The effect of PROX1 silencing (A) and overexpression (B) on the organization of actin cytoskeleton and cell morphology in FTC-133 cells. Following a transient transfection with si PROX1 -RNA or pIRES- PROX1 , the cells were stained with an anti-Prox1 antibody (red), with phalloidin (green) for F-actin and with DAPI (blue) for DNA. The yellow boxes depict areas which are zoomed in the bottom panel.

Article Snippet: Prox1 , Goat anti-human Prox1 (1:2000), R&D System, USA , Affinity-purified rabbit anti-goat antibody/HRP (1:20000), Jackson ImmunoResearch Laboratories, USA.

Techniques: Over Expression, Transfection, Staining

Journal: Oncotarget

Article Title: The role of prospero homeobox 1 (PROX1) expression in follicular thyroid carcinoma cells

doi: 10.18632/oncotarget.23167

Figure Lengend Snippet: The effect of PROX1 depletion on migration (A) , invasion (B) and motility (C) of FTC-133 cells, determined by chamber migration, Matrigel invasion, and wound-healing assay, respectively. Bar graphs illustrate the means ± standard deviation (SD) of the results from four independent experiments. Asterisks ( **** ) denote the P -value < 0.0001. (A) and (B) To evaluate their migration and invasive potential, the FTC-133 cells transfected with si PROX1 or control siNEG RNA were seeded in Boyden insert chambers or Matrigel Invasion Chambers with 8-μm pores. Lower chambers were filled with culture medium supplemented with 10% FBS. After 24 hours, the cells which have passed through membranes were fixed, stained, and photographed at a 40x magnification. (C) The wound closure percentage 24 hours after the scratch in monolayers of the FTC-133 cells transfected with si PROX1 or control siNEG. Representative light microscope images were taken at a 200x magnification.

Article Snippet: Prox1 , Goat anti-human Prox1 (1:2000), R&D System, USA , Affinity-purified rabbit anti-goat antibody/HRP (1:20000), Jackson ImmunoResearch Laboratories, USA.

Techniques: Migration, Wound Healing Assay, Standard Deviation, Transfection, Control, Staining, Light Microscopy

Journal: Oncotarget

Article Title: The role of prospero homeobox 1 (PROX1) expression in follicular thyroid carcinoma cells

doi: 10.18632/oncotarget.23167

Figure Lengend Snippet: The effect of PROX1 overexpression on migration (A) , invasion (B) and motility (C) of FTC-133 cells, determined by chamber migration, Matrigel invasion, and wound-healing assay, respectively. Bar graphs illustrate the means ± standard deviation (SD) of the results from four independent experiments. Asterisks denote the corresponding P -values ( **** : P -value < 0.0001; *** : P -value < 0.01) (A) and (B) To evaluate their migration and invasive potential, the FTC-133 cells transfected pIRES- PROX1 plasmid or empty pIRES plasmid were seeded in Boyden insert chambers or Matrigel Invasion Chambers with 8-μm pores. Lower chambers were filled with culture medium supplemented with 10% FBS. After 24 hours, the cells which have passed through membranes were fixed, stained, and photographed at a 40x magnification. (C) The wound closure percentage 24 hours after the scratch in monolayers of the FTC-133 cells transfected with pIRES- PROX1 or empty pIRES plasmid. Representative light microscope images were taken at a 200x magnification.

Article Snippet: Prox1 , Goat anti-human Prox1 (1:2000), R&D System, USA , Affinity-purified rabbit anti-goat antibody/HRP (1:20000), Jackson ImmunoResearch Laboratories, USA.

Techniques: Over Expression, Migration, Wound Healing Assay, Standard Deviation, Transfection, Plasmid Preparation, Staining, Light Microscopy

Journal: Oncotarget

Article Title: The role of prospero homeobox 1 (PROX1) expression in follicular thyroid carcinoma cells

doi: 10.18632/oncotarget.23167

Figure Lengend Snippet: The effect of the PROX1 knockdown (A) and overexpression (B) in FTC-133 cells on soft agar colony formation. After a transient transfection with si PROX1 or pIRES- PROX1 , the cells were seeded in 6-well culture plates containing soft agar. After two weeks, the colonies were stained and counted under a light microscope at a 100x or 200x magnification. The images show results representative of at least three independent experiments. The areas depicted by yellow boxes are zoomed in the lower panel. Lower panels show quantitative data for soft agar colonies and Matrigel spheroids formed in the two relevant assays. The PROX1 silencing significantly reduced the rate of colony formation, whereas PROX1 overexpression significantly promoted the rate of spheroid formation in FTC-133 cells. Bar graphs illustrate the means ± standard deviation (SD) from five assays, and asterisks ( ** ) indicate the P -value < 0.01.

Article Snippet: Prox1 , Goat anti-human Prox1 (1:2000), R&D System, USA , Affinity-purified rabbit anti-goat antibody/HRP (1:20000), Jackson ImmunoResearch Laboratories, USA.

Techniques: Knockdown, Over Expression, Transfection, Staining, Light Microscopy, Standard Deviation

Journal: Oncotarget

Article Title: The role of prospero homeobox 1 (PROX1) expression in follicular thyroid carcinoma cells

doi: 10.18632/oncotarget.23167

Figure Lengend Snippet: Fixed and permeabilized FTC-133 cells depleted of Prox1 or transfected with siNEG RNA were incubated overnight at 4°C with appropriate primary antibodies, followed by a one-hour room-temperature incubation with the appropriate secondary Rhodamine (TRICT)-conjugated AffiniPure F(ab’) 2 fragment goat anti-rabbit IgG and DyLight549-conjugated AffiniPure goat anti-mouse IgG antibodies (red). Images are representative confocal micrographs of FTC-133/si PROX1 cells in which F-actin was stained with phalloidin-AlexaFluor488 (green) and the nuclei (DNA) with DAPI (blue), taken at a 630x magnification. The yellow boxes depict the zoomed regions in the corresponding images.

Article Snippet: Prox1 , Goat anti-human Prox1 (1:2000), R&D System, USA , Affinity-purified rabbit anti-goat antibody/HRP (1:20000), Jackson ImmunoResearch Laboratories, USA.

Techniques: Transfection, Incubation, Staining

Journal: Oncotarget

Article Title: The role of prospero homeobox 1 (PROX1) expression in follicular thyroid carcinoma cells

doi: 10.18632/oncotarget.23167

Figure Lengend Snippet: The effect of PROX1 knockdown (A) and overexpression (B) on proliferation, cell cycle arrest, and apoptosis in FTC-133 cells. The cell proliferation was measured by the BrdU incorporation assay. Cell cycle profile was evaluated by propidium iodide staining and flow cytometry. Apoptosis rate was measured by propidium iodide and Annexin V-FITC staining followed by flow cytometry. Annexin V-FITC staining discriminates cells in early (lower right quadrant) and advanced (upper right quadrant) apoptotic states. Viable cells are double negative (lower left quadrant).

Article Snippet: Prox1 , Goat anti-human Prox1 (1:2000), R&D System, USA , Affinity-purified rabbit anti-goat antibody/HRP (1:20000), Jackson ImmunoResearch Laboratories, USA.

Techniques: Knockdown, Over Expression, BrdU Incorporation Assay, Staining, Flow Cytometry

Journal: Oncotarget

Article Title: The role of prospero homeobox 1 (PROX1) expression in follicular thyroid carcinoma cells

doi: 10.18632/oncotarget.23167

Figure Lengend Snippet: Primary and secondary antibodies used to analyse the expression of Prox1 and other proteins by western blotting and immunochistochemistry

Article Snippet: Prox1 , Goat anti-human Prox1 (1:2000), R&D System, USA , Affinity-purified rabbit anti-goat antibody/HRP (1:20000), Jackson ImmunoResearch Laboratories, USA.

Techniques: Expressing, Western Blot, Affinity Purification, Purification, Control, Immunofluorescence

Journal: Theranostics

Article Title: Neutrophil extracellular trap-mediated impairment of meningeal lymphatic drainage exacerbates secondary hydrocephalus after intraventricular hemorrhage

doi: 10.7150/thno.91653

Figure Lengend Snippet: NET degradation improves mLV damage. C57BL/6 mice were i.c.v. injected with or without DNase I after IVH. Twenty-four h post-IVH, the meninges were collected for observation. (A) Representative WB images showing the expression of CitH3, VEGFC, LYVE-1, PROX1, FOXC2, and VE-cadherin in the meninges in the Sham, IVH, and IVH+DNase I groups 24 h after IVH. GAPDH was used as an internal reference protein. (B-G) Quantification of CitH3, VEGFC, LYVE-1, PROX1, FOXC2, and VE-cadherin expression (n = 6, one-way ANOVA). (H, I) Relative mRNA levels of FOXC2 and VEGFC in the meninges in the various experimental groups (n = 6). (J, K) The concentrations of VEGFC and FOXC2 in the meninges were quantified by ELISA (n = 6, one-way ANOVA). (L) NO concentrations in the meninges were quantifid in the different groups 24 h after IVH (n = 6, one-way ANOVA). (M) A representative image showing the IF staining of the meninges with anti-LYVE-1 antibodies (red) in the COS. (N) The extent of anti-LYVE-1 antibody staining in the meninges of the COS was quantified and is presented as a percentage of the total area (n = 6, one-way ANOVA). (O) Images showing TUNEL and LYVE-1 staining in the TS of Sham-, IVH-, or DNase I-treated mice were selected as representative examples 24 h after IVH. (P) The number of TUNEL-positive LECs surrounding the TS in the meninges of mice (n = 6, one-way ANOVA). *P < 0.05, **p < 0.01. Data are means ± SEMs.

Article Snippet: The following primary antibodies were added and incubated overnight at 4 °C with gentle agitation: anti-VEGFR3 (goat, 0.1 μg/mL, AF743, R&D), anti-CitH3 (rabbit, 1:2000, ab5103, Abcam), anti-VEGF-C (rabbit, 1:1000, DF7011, Affinity), anti-VE-cadherin (goat, 0.2 μg/mL, AF1002, R&D), anti-PROX1 (rabbit, 1:2000, 11067-2-AP, Proteintech), anti-LYVE-1 (rabbit, 1:1000, 67538S, Cell Signaling Technology), anti-FOXC-2 (rabbit, 1:500, DF3252, Affinity), and anti-CX3CR1 (rabbit, 1:1000, 13885-1-AP, Proteintech).

Techniques: Injection, Expressing, Enzyme-linked Immunosorbent Assay, Staining, TUNEL Assay

Journal: Investigative Ophthalmology & Visual Science

Article Title: A Small Molecule Inhibitor of VE-PTP Activates Tie2 in Schlemm's Canal Increasing Outflow Facility and Reducing Intraocular Pressure

doi: 10.1167/iovs.61.14.12

Figure Lengend Snippet: Expression of VE-PTP and activation of Tie2 by AKB-9778 in SC endothelial cells. ( A ) Adult murine eyes of either wildtype (VE-PTP +/+ ) or heterozygous knock-in mice expressing β-Galactosidase under the VE-PTP promotor (VE-PTP +/mut ) were cross-sectioned after X-Gal staining to visualize VE-PTP expression. ( B ) Sagittal sections of adult murine eyes were stained with antibodies specific for VE-PTP ( green ) or VE-cadherin ( red ). ( C ) Top panel shows labelling of conventional outflow tissues from human donor eyes restricted to the endothelial cells of Schlemm's canal (SC) using antibodies specific for VE-PTP ( violet ). Bottom panel shows selective staining of SC with antibodies against the vascular endothelial cell marker, CD31 ( red ). Sections underwent counterstaining with DAPI for cell nuclei localization. TM = trabecular meshwork, magnification bars = 50 µm. ( D ) Whole mount immunofluorescence analysis of adult murine eyes of VE-PTP +/+ or VE-PTP +/mut mice. Enucleated eyes were stained as whole mounts with X-gal or with antibodies to detect PECAM-1, Prox-1, and Tie2. The entire thickness of the limbus was visualized by confocal imaging and subsets of optical sections containing SC are shown. ( E ) Whole mount immunofluorescence staining of adult murine eyes topically treated with either AKB-9778 or vehicle for 1 hour and stained for PECAM-1, pY992-Tie2, and total Tie2. The entire thickness of the limbus was visualized by confocal imaging and subsets of optical sections depicting SC are shown. Scale bar = 20 µm; SC = Schlemm's canal; AV = aqueous vein; CB = ciliary body; BV = blood vessel; TM = trabecular meshwork.

Article Snippet: The following primary antibodies were used for immunofluorescence staining at the indicated concentrations: VE-PTP (rat-anti-mouse-VE-PTP clone 109.1, 10 µg/mL), (rabbit-anti-human VE-PTP, hVE-PTP 1-8 antibodies were generated in rabbits against a recombinant form of the extracellular fibronectin type III-like domains 1-8 of human VE-PTP and affinity purified using the antigen), VE-cadherin (rabbit-a-mouse-VE-cadherin, pAB42) Prox1 (goat-a-human-Prox1, R&D, AF-2727, 2 µg/mL); PECAM-1 (rat-a-mouse PECAM-1 clone 1 G5.1 (3 µg/mL) + clone 5D2.6 (1 µg/mL), (rabbit polyclonal anti-PECAM-1, Abcam, ab28364); Tie2 (goat-a-mouse-Tie2, R&D, AF762, 5 µg/mL); and phospho-Tie2 (rabbit-a-pY992-Tie2, R&D, AF2720, 10 µg/mL).

Techniques: Expressing, Activation Assay, Knock-In, Staining, Marker, Immunofluorescence, Imaging