Journal: PLoS ONE

Article Title: Efficient Assessment of Developmental, Surgical and Pathological Lymphangiogenesis Using a Lymphatic Reporter Mouse and Its Embryonic Stem Cells

doi: 10.1371/journal.pone.0157126

Figure Lengend Snippet: (A-J) Embryonic tissues: bright field (A) and fluorescence (B) images of the Prox1-tdTomato embryo (E17.5). Distinct lymphatic networks shown in the embryonic skin (C-E). (E) Enlarged image of the boxed area in panel D. Lymphatic vessels in the embryonic liver (F,G) and mesentery membrane (H). Note that hepatocytes (I) and tail nerves (J) are also positive for tdTomato. (K-Y) Adult tissues: headshots of adult wild type and Prox1-tdTomato transgenic mice were taken under a bright light (K), bright and fluorescent light (L) and fluorescent light (M). Lymphatic vessels were easily detectable in various tissues such as the ear (N), eye (O,P), tail (Q), tongue (R), trachea (S), diaphragm muscle (T), bladder (U,V), intestine (W), mesentery (X) and lymph node (Y). Corneal limbal lymphatic (arrow) and Schlemm’s canal (arrowhead) of the eye were shown in two consecutive focal planes (O,P). Bilateral lymphatic collectors in the tail were marked with two arrows (Q).

Article Snippet: Prox1 staining was performed using anti-Prox1 antibody (R&D Systems).

Techniques: Fluorescence, Membrane, Transgenic Assay

Journal: PLoS ONE

Article Title: Efficient Assessment of Developmental, Surgical and Pathological Lymphangiogenesis Using a Lymphatic Reporter Mouse and Its Embryonic Stem Cells

doi: 10.1371/journal.pone.0157126

Figure Lengend Snippet: (A-H) Axillary lymphadenectomy model: Fluorescent light enables the researchers to easily locate the axillary lymph node (A-D). From a different set of mice, the left-side axillary lymph nodes were removed, while the right-side nodes were left intact. To enhance lymphatic growth in the surgical site, 9-cis RA was treated for the first 5 days after surgery. After 4 weeks, lymphatic vessels were visualized in the left vs . right side of the armpit of the mice with asterisks indicating the original locations of the lymph nodes (E,F). Images in the boxed areas (E, F) are shown in panels G and H. (I-Q) Lymph node transplantation model: the axillary lymph node in Prox1-tdTomato mice (recipient) was replaced with the matching lymph node from Prox1-EGFP mice (donor), followed by administration of 9-cis RA for 4 weeks. Panels O, P and Q are enlarged images of the boxed areas in panels K, M and N, respectively. (R-U) Tail lymphedema model: representative images of the circumcised tails at post-operational days 1 and 15 are shown (R,T). At both days, the circumcised tail skins were prepared inside-out to visualize dermal lymphatic vessels (S,U). Asterisks mark the wounded area and black dotted lines indicate the wound border. Note the numerous newly formed lymphatics invading into the healed area (S).

Article Snippet: Prox1 staining was performed using anti-Prox1 antibody (R&D Systems).

Techniques: Transplantation Assay

Journal: PLoS ONE

Article Title: Efficient Assessment of Developmental, Surgical and Pathological Lymphangiogenesis Using a Lymphatic Reporter Mouse and Its Embryonic Stem Cells

doi: 10.1371/journal.pone.0157126

Figure Lengend Snippet: (A-E) Bladder lymphangiogenesis model: Adult Prox1-tdTomato mice were i.p. injected with phosphate-buffered saline (A,C) or cyclophosphamide (B,D) at days 1 and 4. At day 9, lymphatic vessels in their bladders were visualized. Morphometric analyses revealed a significantly increase in the number of branch (marked with arrowhead), loop (arrow) and blind ends (double arrowhead) of lymphatic vessels in the CYP-treated bladder compared to the PBS-treated control (CTR) bladder (E). (F-J) Tumor implantation model: GFP-labeled tumor cells were implanted into the skin of immunodeficient athymic Prox1-tdTomato mouse, generated by crossing athymic nude mice and Prox1-tdTomato mice. Images of the GFP-labeled tumor mass (F), dermal lymphatic in the surrounding tissues (G) and their merged image (H) are shown. (I) and (J) are enlarged images of the boxed areas in panel (H) and (I), respectively. Arrows mark luminal valves.

Article Snippet: Prox1 staining was performed using anti-Prox1 antibody (R&D Systems).

Techniques: Injection, Saline, Control, Tumor Implantation, Labeling, Generated

Journal: PLoS ONE

Article Title: Efficient Assessment of Developmental, Surgical and Pathological Lymphangiogenesis Using a Lymphatic Reporter Mouse and Its Embryonic Stem Cells

doi: 10.1371/journal.pone.0157126

Figure Lengend Snippet: (A,B) Bright field (BF) and fluorescent (UV) images of the head of Prox1-tdTomato, Prox1-tdTomato/Prox1-EGFP and Prox1-EGFP lymphatic-specific reporter mice. (C-K) Expressions of tdTomato and/or EGFP proteins were visualized in the skin of embryos (EM, C-E) and new pups (P1, F-H), or in the bladder of adult (BL, I-J) of each genotype. (L-N) Prox1 whole-mount staining of the ear of tdTomato adult mouse.

Article Snippet: Prox1 staining was performed using anti-Prox1 antibody (R&D Systems).

Techniques: 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: 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: Cell reports

Article Title: Early Seizure Activity Accelerates Depletion of Hippocampal Neural Stem Cells and Impairs Spatial Discrimination in an Alzheimer’s Disease Model

doi: 10.1016/j.celrep.2019.05.101

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Mouse anti-Prox1 , PhosphoSolutions , Cat#: 1685-Prox1; RRID: AB_2492217.

Techniques: Recombinant, Imaging, Software, Microscopy