Journal: Biology of reproduction

Article Title: Immunolocalization of acidic and basic fibroblast growth factors in porcine uterine and conceptus tissues.

doi: 10.1095/biolreprod56.6.1527

Figure Lengend Snippet: FIG. 1. Western blot analysis of porcine uterine protein extract at Day 12 of gestation. a) Molecular weight markers (x 10 3) (lane 1), total CM-50 sephadex-extracted uterine protein (25 g, lanes 2, 4, and 6), human recombinant bFGF (100 ng, lanes 3 and 5). Lanes 1 and 2 were stained for total protein by colloidal gold stain. Lanes 3 and 4 were in- cubated with anti-bFGF antibody (0.15 Ixg/ml). Lanes 5 and 6 were in- cubated with rabbit IgG (0.15 .Lg/ml). b) Sephadex-extracted uterine pro- tein (25 jIg, stained for total protein by colloidal gold) in nonreducing 7.5-15% PAGE. Bovine aFGF (500 ng, lanes 2, 4, and 6) and sephadex extract (25 g, lanes 3, 5, and 7). Immunoreactive aFGF bands are ob- served in lanes 2 and 3 (incubated with anti-aFGF antibody). The absence of these bands is noted in lanes 4 and 5 (anti-aFGF antibody immunoab- sorbed with bovine aFGF) and in lanes 6 and 7 (incubated with rabbit IgG).

Article Snippet: For aFGE PAGE was performed using 7.5-15% acrylamide gel and bovine aFGF (cat. #132-FA; R&D Systems, Minneapolis, MN) as standard.

Techniques: Western Blot, Molecular Weight, Recombinant, Staining, Incubation

Journal: The Journal of Cell Biology

Article Title: Septin2 mediates podosome maturation and endothelial cell invasion associated with angiogenesis

doi: 10.1083/jcb.201903023

Figure Lengend Snippet: Septin2 localization following Src activation. ECs expressing RapR-Src-Cerulean (magenta, top left) and Septin2-YFP fusion protein (green, top right) were imaged live following Src activation. The white arrow in the overlay (bottom left) indicates the colocalization of both Septin2 and Src to rosette structure. Time of RapR-Src activation is indicated by the “Rapamycin Added” caption. The video accompanies Fig. S1, B and C, and is displayed at 0.5 s per frame.

Article Snippet: Chemical reagents used were as follows: Alexa Fluor 647 phalloidin (Cat# A22287; Thermo Fisher), Alexa Fluor plus 405 phalloidin (Cat# A30104; Thermo Fisher), D- erythro -sphingosine-1-phosphate (Cat# 860492; Avanti Polar Lipids), normal donkey serum (Cat# 017–000-121; Jackson Immunoresearch), rapamycin (Cat# R5000; LC Laboratories), puromycin dihydrochloride (Cat# P-600-100; Gold Biotechnology), insulin (Cat# I-1882; Sigma-Aldrich), human holo transferrin (Cat# T-4132; Sigma-Aldrich), sodium selenite (Cat# 5–5261; Sigma-Aldrich), FA-free BSA (Cat# A8806; Sigma-Aldrich), oleic acid (Cat# O7501; Sigma-Aldrich), collagen I, bovine (Cat# A10644-01; GIBCO BRL), Alexa Fluor 405 NHS Ester (succinimidyl ester; Cat# A30000; Thermo Fisher), Oregon Green-488–conjugated gelatin (Cat# G13186; Thermo Fisher), ascorbic acid (Cat# A4034; Sigma-Aldrich), and basic fibroblast growth factor (Cat# 234-FSE; R&D Systems), VEGF165, human (Cat# 1150–05-10; Goldbiotech), GM6001 MMP Inhibitor (Cat# CC1000; Sigma-Aldrich), MT1-MMP inhibitor, NSC405020 (Cat# 444295; Sigma-Aldrich), blebbistatin (Cat# 2406–1; BioVision), and saracatinib (Cat# sc-45364607; Santa Cruz).

Techniques: Activation Assay, Expressing

Journal: The Journal of Cell Biology

Article Title: Septin2 mediates podosome maturation and endothelial cell invasion associated with angiogenesis

doi: 10.1083/jcb.201903023

Figure Lengend Snippet: Septin2 expression is required for podosome-mediated matrix degradation and invasion in vitro. (A–D) Effect of Septin2 downregulation on the ability of ECs to form functional podosomes in response to Src activation. HPAECs were treated with 35 nM siRNA targeting Septin2 (SEPT2) or NT scramble control for 48 h before being seeded onto Alexa488-gelatin and transduced with adenoviral constructs expressing RapR-Src and FRB. After an additional 24 h, RapR-Src was activated for 30 min, 1 h, and 2 h with rapamycin (500 nm), and cells were fixed and stained for F-actin. (A) Representative epifluorescence images of HPAECs 30 min following Src activation. White arrows indicate functional podosomes defined as in (scale bar = 50 µm). (B) Percentage of HPAECs displaying the matrix-degrading podosome phenotype before (0 h) and after activation of Src (time points are indicated). For each condition, a minimum of 10 fields of view were analyzed per experiment. Data show results from three independent experiments. KD, knockdown. (C) Western blot analysis displays downregulation of endogenous Septin2 with respect to GAPDH loading control used in A. (D) Average relative Septin2 expression normalized to scramble control ( n = 3). Error bars represent standard deviation. (E) Role of Septin2 in VEGF-stimulated matrix degradation. Septin2 expression was downregulated as in A. Cells were deprived of VEGF for 48 h, serum starved in for 2 h, and then seeded onto Alexa405-gelatin. Total matrix degradation was measured after 5 h of VEGF stimulation. At least 10 fields of view per condition were analyzed from three independent experiments. Individual data points are shown around mean value ± SE (whiskers). ( F and G) Septin2 localizes to invading sprouts in vitro. (F) HPAECs were seeded atop a 3D collagen matrix and stimulated using growth factor/S1P mix for 20 h. Following stimulation, cells were fixed and stained for endogenous Septin2 and MT1-MMP (confocal images, scale bar = 10 µm). (G) Representative 3D surface rendering of an invading sprout shown in F created with Imaris (indicated by white box; DAPI-stained nuclei represented in blue). (H and I) Effect of Septin2 downregulation on cell invasion. Septin2 expression was downregulated as in A and invasion was stimulated as in F. Following stimulation, cells were fixed and stained with toluidine blue. (H) Representative images showing cell invasion (lateral view of invading sprouts; scale bar = 0.15 mm). (I) Quantification of invading sprouts. Each data point represents an average of two to four fields per well imaged over three independent experiments. Data are represented as mean ± SE. Significance was determined using a two-sample t test (comparisons indicated by lines; B, E, and I).

Article Snippet: Chemical reagents used were as follows: Alexa Fluor 647 phalloidin (Cat# A22287; Thermo Fisher), Alexa Fluor plus 405 phalloidin (Cat# A30104; Thermo Fisher), D- erythro -sphingosine-1-phosphate (Cat# 860492; Avanti Polar Lipids), normal donkey serum (Cat# 017–000-121; Jackson Immunoresearch), rapamycin (Cat# R5000; LC Laboratories), puromycin dihydrochloride (Cat# P-600-100; Gold Biotechnology), insulin (Cat# I-1882; Sigma-Aldrich), human holo transferrin (Cat# T-4132; Sigma-Aldrich), sodium selenite (Cat# 5–5261; Sigma-Aldrich), FA-free BSA (Cat# A8806; Sigma-Aldrich), oleic acid (Cat# O7501; Sigma-Aldrich), collagen I, bovine (Cat# A10644-01; GIBCO BRL), Alexa Fluor 405 NHS Ester (succinimidyl ester; Cat# A30000; Thermo Fisher), Oregon Green-488–conjugated gelatin (Cat# G13186; Thermo Fisher), ascorbic acid (Cat# A4034; Sigma-Aldrich), and basic fibroblast growth factor (Cat# 234-FSE; R&D Systems), VEGF165, human (Cat# 1150–05-10; Goldbiotech), GM6001 MMP Inhibitor (Cat# CC1000; Sigma-Aldrich), MT1-MMP inhibitor, NSC405020 (Cat# 444295; Sigma-Aldrich), blebbistatin (Cat# 2406–1; BioVision), and saracatinib (Cat# sc-45364607; Santa Cruz).

Techniques: Expressing, In Vitro, Functional Assay, Activation Assay, Control, Transduction, Construct, Staining, Knockdown, Western Blot, Standard Deviation

Journal: Frontiers in Pharmacology

Article Title: Oleoylethanolamide as a New Therapeutic Strategy to Alleviate Doxorubicin-Induced Cardiotoxicity

doi: 10.3389/fphar.2022.863322

Figure Lengend Snippet: OEA inhibited DOX-induced oxidative stress and apoptosis in cardiomyocytes through TRPV1 pathway. HL-1 cells were treated with 0.1% DMSO, OEA (30 μM), GW6471 (5 μM), capsazepine (10 μM), nonivamide (10 μM), fenofibrate (100 μM) for 30 min. Cells were then treated with DOX (2 μM) for 24 h. Levels of (A) GST (B) TBARS and (C) cell viability was assessed in HL-1 cardiomyocytes. (D) TUNEL positive cells shown in (E) were counted. (E) TUNEL staining of HL-1 cardiomyocytes. Data are expressed as mean ± SEM, n = 4. ***, p < 0.001 vs. control. ##, p < 0.01; ###, p < 0.001 vs. DOX + vehicle. $$$, p < 0.001 vs. DOX + OEA.

Article Snippet: Adult derived primary human cardiomyocytes (Celprogen, Cat # 36044-15) were cultured in complete growth media (Celprogen, Cat #M36044-15S) in an incubator at 37°C with 5% CO 2 atmosphere.

Techniques: TUNEL Assay, Staining

Journal: Frontiers in Pharmacology

Article Title: Oleoylethanolamide as a New Therapeutic Strategy to Alleviate Doxorubicin-Induced Cardiotoxicity

doi: 10.3389/fphar.2022.863322

Figure Lengend Snippet: OEA promotes TRPV1-mediated PI3K/ Akt signaling in cardiomyocytes. HL-1 cells were treated with 0.1% DMSO, OEA (30 μM), GW6471 (5 μM), capsazepine (10 μM) and nonivamide (10 μM) for 30 min. Cells were then treated with DOX (2 μM) for 24 h. PI3K, Akt, p-PI3K and p-Akt was determined using western blot analysis.

Article Snippet: Adult derived primary human cardiomyocytes (Celprogen, Cat # 36044-15) were cultured in complete growth media (Celprogen, Cat #M36044-15S) in an incubator at 37°C with 5% CO 2 atmosphere.

Techniques: Western Blot

Journal: Frontiers in Pharmacology

Article Title: Oleoylethanolamide as a New Therapeutic Strategy to Alleviate Doxorubicin-Induced Cardiotoxicity

doi: 10.3389/fphar.2022.863322

Figure Lengend Snippet: Inhibition of the PI3K-Akt signaling blocks the protective effects of OEA in cardiomyocytes. HL-1 cells were treated with 0.1% DMSO, OEA (30 μM) and LY294002 (10 μM) for 30 min. Cells were then treated with DOX (2 μM) for 24 h. Levels of (A) GST (B) TBARS and (C) cell viability was assessed in HL-1 cardiomyocytes. (D) TUNEL positive cells shown in (F) were counted. (E) Fluorescence quantification for caspase three in (G) were calculated. (F) TUNEL staining of HL-1 cardiomyocytes. (G) Immunofluorescence staining of caspase three in HL-1 cardiomyocytes. Data are expressed as mean ± SEM, n = 4. ***, p < 0.001 vs. control. #, p < 0.05; ##, p < 0.01; ###, p < 0.001 vs. DOX + vehicle. $, p < 0.05; $$, p < 0.01; $$$, p < 0.001 vs. DOX + OEA.

Article Snippet: Adult derived primary human cardiomyocytes (Celprogen, Cat # 36044-15) were cultured in complete growth media (Celprogen, Cat #M36044-15S) in an incubator at 37°C with 5% CO 2 atmosphere.

Techniques: Inhibition, TUNEL Assay, Fluorescence, Staining, Immunofluorescence