Journal: PloS one

Article Title: New candidate biomarkers in the female genital tract to evaluate microbicide toxicity.

doi: 10.1371/journal.pone.0110980

Figure Lengend Snippet: Figure 1. Quantitative proteomic analysis of mouse brush samples. (A) LC-MS/MS analysis of mouse proteins in vaginal brush samples that are affected by treatment with N9 (open, black circles) or BZK (closed squares). Shown are proteins that are increased compared to vehicle controls. The following proteins are depicted in the graph: fibrinogen alpha polypeptide isoform 2 (FGA), plasminogen (PLG), apolipoprotein A-1 (Apo A-1), and apolipoprotein C-1 (Apo C-1). Each data point represents the average of three replicates per individual experimental study. Only proteins shown to change in four studies are represented. (B) ELISA analysis of mouse proteins in vaginal brush samples that are increased in response to treatment with 8% N9 or 2% BZK. Statistical analysis was performed using a two-tailed Student’s T-test of N9 and BZK samples compared to vehicle controls (**** = p#0.0001, ** = p#0.01). (C) ELISA analysis of mouse proteins in vaginal brush samples that are increased in response to treatment with vehicle, 1% N9, 4% N9, or 8% N9. Statistical analysis was performed using a two-tailed student’s t-test of N9 and BZK samples compared to vehicle controls (**** = p#0.0001, *** = p#0.001, ** = p#0.01, * = p#0.05, NS = not significant). doi:10.1371/journal.pone.0110980.g001

Article Snippet: Reagents Nonoxynol-9 (N9) was purchased from Spectrum Chemicals and Laboratory Products (Gardena, CA); tenofovir was purchased from AK Scientific (Union City, CA); benzalkonium chloride (BZK) and high viscosity sodium carboxymethylcellulose (CMC) were purchased from Sigma Aldrich (Saint Louis, MO); mouse and rabbit apolipoprotein A-I, apolipoprotein C-1, CD166, and rabbit IL-8 ELISA kits were purchased from EIAab (Wuhan, China); mouse fibrinogen ELISA kit was purchased from Abcam (Cambridge, MA); mouse plasminogen ELISA kit was purchased from GenWay (San Diego, CA); mouse IL-8 ELISA kit was purchased from MyBioSource (San Diego, CA); rabbit plasminogen ELISA kit was purchased from Alpco (Salem, NH); rabbit fibrinogen ELISA kit was purchased from Molecular Innovations (Novi, MI); medroxyprogesteron acetate was purchased from Pharmacia (New York, NY); CellTiter-Glo was purchased from Promega (Madison, WI); RNeasy kits were purchased from Qiagen (Valencia, CA); rabbit anti-human olfactomedin 4 (OLFM-4) polyclonal antibody was purchased from Abcam (Cambridge, MA); rabbit anti-human mucin 5B polyclonal antibody was purchased from Bioss USA Antibodies (Woburn, MA); rabbit anti-mouse CD166 was purchased from GeneTex (Irvine, CA); rabbit anti-mouse b-actin was purchased from Abcam (Cambridge, MA); IRDye 800 Goat anti-rabbit IgG was purchased from Li-Cor (Lincoln, NE); mouse anti-human b-actin monoclonal antibody was purchased from Cell Signaling Technology (Danvers, MA); donkey anti-mouse IgG antibody conjugated to IRDye 680 LT and goat anti-rabbit IgG antibody conjugated to IRDye 800CW were purchased from LI-COR biosciences (Lincoln, NE).

Techniques: Liquid Chromatography with Mass Spectroscopy, Enzyme-linked Immunosorbent Assay, Two Tailed Test

Journal: PloS one

Article Title: New candidate biomarkers in the female genital tract to evaluate microbicide toxicity.

doi: 10.1371/journal.pone.0110980

Figure Lengend Snippet: Figure 5. ELISA analysis of rabbit proteins in vaginal brush samples increased in response to treatment with N9 or BZK. Statistical analysis were performed using a two-tailed Student’s T-test of N9 and BZK samples compared to vehicle controls (** = p#0.01, * = p# 0.05, NS = not significant). doi:10.1371/journal.pone.0110980.g005

Article Snippet: Reagents Nonoxynol-9 (N9) was purchased from Spectrum Chemicals and Laboratory Products (Gardena, CA); tenofovir was purchased from AK Scientific (Union City, CA); benzalkonium chloride (BZK) and high viscosity sodium carboxymethylcellulose (CMC) were purchased from Sigma Aldrich (Saint Louis, MO); mouse and rabbit apolipoprotein A-I, apolipoprotein C-1, CD166, and rabbit IL-8 ELISA kits were purchased from EIAab (Wuhan, China); mouse fibrinogen ELISA kit was purchased from Abcam (Cambridge, MA); mouse plasminogen ELISA kit was purchased from GenWay (San Diego, CA); mouse IL-8 ELISA kit was purchased from MyBioSource (San Diego, CA); rabbit plasminogen ELISA kit was purchased from Alpco (Salem, NH); rabbit fibrinogen ELISA kit was purchased from Molecular Innovations (Novi, MI); medroxyprogesteron acetate was purchased from Pharmacia (New York, NY); CellTiter-Glo was purchased from Promega (Madison, WI); RNeasy kits were purchased from Qiagen (Valencia, CA); rabbit anti-human olfactomedin 4 (OLFM-4) polyclonal antibody was purchased from Abcam (Cambridge, MA); rabbit anti-human mucin 5B polyclonal antibody was purchased from Bioss USA Antibodies (Woburn, MA); rabbit anti-mouse CD166 was purchased from GeneTex (Irvine, CA); rabbit anti-mouse b-actin was purchased from Abcam (Cambridge, MA); IRDye 800 Goat anti-rabbit IgG was purchased from Li-Cor (Lincoln, NE); mouse anti-human b-actin monoclonal antibody was purchased from Cell Signaling Technology (Danvers, MA); donkey anti-mouse IgG antibody conjugated to IRDye 680 LT and goat anti-rabbit IgG antibody conjugated to IRDye 800CW were purchased from LI-COR biosciences (Lincoln, NE).

Techniques: Enzyme-linked Immunosorbent Assay, Two Tailed Test

Journal: Diabetes

Article Title: Plasminogen Activator Inhibitor-1 Is Involved in Streptozotocin-Induced Bone Loss in Female Mice

doi: 10.2337/db12-1552

Figure Lengend Snippet: Effects of PAI-1 deficiency on diabetic bone loss in both sexes of mice. Hematoxylin-eosin staining of tibia in control and streptozotocin-treated male and female PAI-1 WT and KO mice ( A ). BMD values in total, trabecular, and cortical bones ( B ); cortical thickness ( C ); and second moment of minimum and polar areas ( D ) of tibia in control and streptozotocin-treated male PAI-1 WT and KO mice. BMD values in total, trabecular, and cortical bones ( E ); cortical thickness ( F ); and second moment of minimum and polar areas ( G ) of tibia in control and streptozotocin-treated female PAI-1 WT and KO mice. For assessment of trabecular BMD, trabecular regions of interest extended from 96 um distal to the end of the proximal growth plate over 1.5 mm toward the diaphysis. For assessment of cortical BMD and thickness, cortical ROIs were defined as 2.0-mm segments of the mid-diaphysis tibia. For assessment of total BMD and bone strength index (second moment of minimum and polar areas: index of bending strength), ROIs were defined as 9,600-μm segment (100 slices) from distal end of proximal growth plate of tibia. Parameters used for the CT scans were as follows: tube voltage, 50 kVp; tube current, 500 μA; integration time, 3.6 ms; axial field of view, 48 mm; and voxel size of 48 × 96 μm with a slice thickness of 96 μm. Bone parameters were analyzed using the LaTheta software (version 3.40). Results are expressed as means ± SEM. * P < 0.05, ** P < 0.01 ( n = 5–7 in each group). Cont, control; STZ, streptozotocin.

Article Snippet: Plasma total PAI-1 was measured using a Murine Total PAI-1 ELISA kit (Molecular Innovations, Novi, MI).

Techniques: Staining, Software

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