Journal: The Journal of Neuroscience

Article Title: Urokinase-Type Plasminogen Activator Promotes Dendritic Spine Recovery and Improves Neurological Outcome Following Ischemic Stroke

doi: 10.1523/JNEUROSCI.5309-13.2014

Figure Lengend Snippet: uPA induces cytoskeletal reorganization in dendritic protrusions. A, Representative micrograph of MAP-2 (green) and phalloidin (white and red) staining in a dendrite of a Wt cerebral cortical neuron incubated 60 min with either 10 nm of uPA (+ uPA) or vehicle (control). Arrows in e denote phalloidin-positive dendritic filopodia. Magnification: 60×. B, Representative micrograph of MAP-2 (green) and profilin (white and red) staining in a Wt cerebral cortical neuron incubated 60 min with either 10 nm of uPA (+ uPA) or vehicle (control). Note that uPA treatment induces the expression of profilin not only in the dendrite but also in the axon. Magnification: 60×. C–G, Representative Western bot analysis and quantification of the mean density of the band of profilin (C) and cofilin (D, E) phosphorylated at serine 3 (p-cofilin) in Wt cerebral cortical neurons incubated 0–60 min with 10 nm uPA (C, D), or 10 nm of uPA's N-terminal fragment (ATF; E), or a combination of uPA and either 5 μm of the Rac inhibitor EHT-1864 (Rac-I; F), or 10 μm of the ROCK inhibitor Y-27632 (ROCK-I; G). C, D, *p < 0.0001 and **p < 0.0001 compared with cells treated 0–5 min with uPA. E, *p < 0.0001 and **p < 0.0001 compared with cells either left untreated or incubated 30 min with uPA or ATF. F, *p < 0.0001 and **p < 0.0001 compared with cells treated with uPA in the presence of the Rac inhibitor EHT-1864. Statistical analysis was performed with one-way ANOVA.

Article Snippet: Experiments were approved by the Institutional Animal Care and Use Committee of Emory University, Atlanta, GA. Recombinant murine uPA and uPA's N-terminal fragment (ATF) were purchased from Molecular Innovations.

Techniques: Staining, Incubation, Expressing, Western Blot

Journal: Journal of medicinal chemistry

Article Title: 6-Substituted Hexamethylene Amiloride (HMA) Derivatives as Potent and Selective Inhibitors of the Human Urokinase Plasminogen Activator for Use in Cancer

doi: 10.1021/acs.jmedchem.8b00838

Figure Lengend Snippet: Inhibition of low molecular weight human uPA by 18 (black) and 27 (red). Data points represent the mean ± SEM (n = 3) from a single representative experiment.

Article Snippet: Experiments with Mouse uPA (active mouse urokinase, HMW, Molecular Innovations Inc., MI, USA) were performed as above with a final enzyme concentration of 20 nM (K m = 580 μ M).

Techniques: Inhibition, Molecular Weight

Journal: Journal of medicinal chemistry

Article Title: 6-Substituted Hexamethylene Amiloride (HMA) Derivatives as Potent and Selective Inhibitors of the Human Urokinase Plasminogen Activator for Use in Cancer

doi: 10.1021/acs.jmedchem.8b00838

Figure Lengend Snippet: Activities of Compounds 4, 5, 18, and 26 against Human and Mouse uPA

Article Snippet: Experiments with Mouse uPA (active mouse urokinase, HMW, Molecular Innovations Inc., MI, USA) were performed as above with a final enzyme concentration of 20 nM (K m = 580 μ M).

Techniques:

Journal: Circulation

Article Title: Releasing the Brakes on the Fibrinolytic System in Pulmonary Emboli: Unique Effects of Plasminogen Activation and α2-Antiplasmin Inactivation

doi: 10.1161/CIRCULATIONAHA.116.024421

Figure Lengend Snippet: Lung tissue containing FITC-fibrin labeled (green) pulmonary emboli was immunostained (red) to detect the expression of (A) tPA, (B) uPA and (C) plasminogen (Pg) (D) The total immune-stained area (arbitrary units; a.u) for each protein was measured in a 20× (100 μm) image of an embolized vs non embolized (Control; dashed yellow outline) pulmonary artery in the lungs. n=3, mean ± SEM. **p<0.01; ns, non-significant.

Article Snippet: The primary antibodies include rabbit anti-mouse against tPA (ASMTPA-GF; Molecular Innovations), uPA (urokinase plasminogen activator) (ASMUPA-GF-HT; Molecular Innovations) and PAI-1 (IASMPAI-GF; Innovative Research), rabbit anti-mouse plasminogen (Abcam), goat anti-mouse α2-antiplasmin (AF1239; R&D Systems), and rat anti-mouse Ly6G (clone1A8, #127602; Biolegend).

Techniques: Labeling, Expressing, Staining

Journal: Frontiers in Pharmacology

Article Title: Ulinastatin Inhibits Osteoclastogenesis and Suppresses Ovariectomy-Induced Bone Loss by Downregulating uPAR

doi: 10.3389/fphar.2018.01016

Figure Lengend Snippet: Ulinastatin inhibits the formation of osteoclasts and bone loss in OVX mice, meanwhile decreases the serum level of uPAR. (A) Distal femurs were embedded with paraffin and sliced up, then H&E and TRAP staining were performed. (B,C) Distal femurs were embedded with paraffin and sliced up, then TRAP staining were performed. The osteoclast number/bone surface (N.Oc/BS, N/mm) was quantified. Data are represented as mean ± SD. n = 10 and ∗ P < 0.05. (D) The serum level of uPAR was detected by ELISA. Data are presented as mean ± SD. n = 10 and ∗ P < 0.05.

Article Snippet: The serum level of uPAR was evaluated by mouse uPAR ELISA kit (Boster, Wuhan, China).

Techniques: Staining, Enzyme-linked Immunosorbent Assay

Journal: Frontiers in Pharmacology

Article Title: Ulinastatin Inhibits Osteoclastogenesis and Suppresses Ovariectomy-Induced Bone Loss by Downregulating uPAR

doi: 10.3389/fphar.2018.01016

Figure Lengend Snippet: Ulinastatin reduces the expression of uPAR, NFATc1 and osteoclast marker genes induced by RANKL. C represents control group, R represents RANKL group, R + ulinastatin represents RANKL + 800 units/mL ulinastatin group. (A) Ulinastatin reduces RANKL-induced mRNA expression of cathepsin K, Trap, Rank, NFATc1, and uPAR. BMMs were cultured with M-CSF (30 ng/mL) and RANKL (100 ng/mL), and treated with or without ulinastatin (800 units/mL) for 4 days. mRNA expression was detected by qRT-PCR. The qRT-PCR experiments have been repeated with different RNA preparations for 3 times independently. Data are represented as mean ± SD . ∗ P < 0.05 and ∗∗ P < 0.01. (B,C,D) Ulinastatin reduces RANKL-induced protein expression of uPAR, cathepsin K and Trap. BMMs were cultured with M-CSF (30 ng/mL) and RANKL (100 ng/mL), and treated with or without ulinastatin (800 units/mL) for 2 or 4 days. Protein expression levels of uPAR, cathepsin K and Trap were examined by western blotting at the indicated times. The amount of loaded protein was 25 μg. The experiment was performed three times independently and GAPDH was used as a loading control. ∗ P < 0.05, ∗∗ P < 0.01 versus RANKL group.

Article Snippet: The serum level of uPAR was evaluated by mouse uPAR ELISA kit (Boster, Wuhan, China).

Techniques: Expressing, Marker, Control, Cell Culture, Quantitative RT-PCR, Western Blot

Journal: Frontiers in Pharmacology

Article Title: Ulinastatin Inhibits Osteoclastogenesis and Suppresses Ovariectomy-Induced Bone Loss by Downregulating uPAR

doi: 10.3389/fphar.2018.01016

Figure Lengend Snippet: Ulinastatin decreased actin ring formations and suppressed uPAR expression. C represents control group, R represents RANKL group, 100 ∼ 800 represents RANKL + 100 units/mL ulinastatin ∼ RANKL + 800 units/mL ulinastatin group. BMMs (1 × 10 4 cells/well) were cultured with M-CSF (30 ng/mL) and RANKL (100 ng/mL), treated with four different concentrations of ulinastatin (100, 200, 400 and 800 units/mL) for 4 days. Then the cells were stained for actin ring assay and Immunofluorescence assay. Images were obtained by fluorescence microscopy. The experiment was performed three times independently.

Article Snippet: The serum level of uPAR was evaluated by mouse uPAR ELISA kit (Boster, Wuhan, China).

Techniques: Expressing, Control, Cell Culture, Staining, Immunofluorescence, Fluorescence, Microscopy

Journal: Frontiers in Pharmacology

Article Title: Ulinastatin Inhibits Osteoclastogenesis and Suppresses Ovariectomy-Induced Bone Loss by Downregulating uPAR

doi: 10.3389/fphar.2018.01016

Figure Lengend Snippet: Knockdown of uPAR decreased RANKL-induced osteoclast and actin ring formation. NC represents siRNA negative control, Si-1, Si-2, and Si-3 represent siRNAs that silence three different fragment of uPAR. BMMs were seeded in 96-well plates and transfected with three siRNAs and NC siRNA, respectively, and cultured with M-CSF (30 ng/mL) and RANKL (50 ng/mL) for 5 days. (A) Immunofluorescence and (B) TRAP staining was performed then. The experiment was performed three times independently.

Article Snippet: The serum level of uPAR was evaluated by mouse uPAR ELISA kit (Boster, Wuhan, China).

Techniques: Knockdown, Negative Control, Transfection, Cell Culture, Immunofluorescence, Staining