Journal: International Journal of Molecular Sciences

Article Title: In Vitro Effects of Papaverine on Cell Migration and Vascular Endothelial Growth Factor in Cancer Cell Lines

doi: 10.3390/ijms23094654

Figure Lengend Snippet: The relative percentage migration compared to MDA-MB-231 ( A ), A549 ( B ), and DU145 ( C ) cells at 0 h exposure when cells were exposed to PPV (10–150 µM). The average of three independent experiments is represented by the graph with error bars indicating standard deviation. The blue bar represents the percentage of the cell migration of the cells after 18 h of exposure, the orange bar represents the percentage of cell migration of cells after 24 h of exposure, and the grey bar represents the percentage of cell migration of cells after 48 h of exposure. The statistical significance of cell migration after 18 h of exposure is indicated with an asterisk (**), the statistical significance of cell migration after 24 h of exposure is indicated with two asterisks (***), and the statistical significance of cell migration after 48 h of exposure indicated with three asterisks (*). The positive control for the migration assay-included cells exposed to 2-ethyl-17-hydroxy-13-methyl-7,8,9,11,12,13,14,15,16,17-decahydro-6-cyclopenta[a]phenanthren-3-yl sulphamate (ESE-ol) (0.4 µM) for 48 h. Statistical significance is represented by an * when using the student t -test with a p value of 0.05 compared to cells propagated in complete growth medium.

Article Snippet: The effects of PPV on the expression of VEGF ligand B and receptors 1 and 2 were investigated in MDA-MB-231, A549, and DU145 cells using a VEGF B ELISA kit (Elabscience biotechnology incorporated (Houston, TX, USA)) a VEGF R1 ELISA kit and a VEGF R2 ELISA kit (Abcam plc. (Cambridge, UK).

Techniques: Migration, Standard Deviation, Positive Control

Journal: International Journal of Molecular Sciences

Article Title: In Vitro Effects of Papaverine on Cell Migration and Vascular Endothelial Growth Factor in Cancer Cell Lines

doi: 10.3390/ijms23094654

Figure Lengend Snippet: Spectrophotometry results of VEGF B ELISA demonstrating the effects of PPV (10–300 µM) on proliferation on MDA-MB-231 cells compared to A549- and DU145 cell lines at 48 h. The average of three independent experiments is represented by the graph with error bars indicating standard deviation. The statistical significance of MDA-MB-231 cells is indicated with an asterisk (*), the statistical significance of A549 cells is indicated with two asterisks (**), and the statistical significance of DU145 cells is indicated with three asterisks (***). Statistical significance is represented by an * when using the student t -test with a p value of 0.05 compared to cells propagated in complete growth medium.

Article Snippet: The effects of PPV on the expression of VEGF ligand B and receptors 1 and 2 were investigated in MDA-MB-231, A549, and DU145 cells using a VEGF B ELISA kit (Elabscience biotechnology incorporated (Houston, TX, USA)) a VEGF R1 ELISA kit and a VEGF R2 ELISA kit (Abcam plc. (Cambridge, UK).

Techniques: Spectrophotometry, Enzyme-linked Immunosorbent Assay, Standard Deviation

Journal: International Journal of Molecular Sciences

Article Title: In Vitro Effects of Papaverine on Cell Migration and Vascular Endothelial Growth Factor in Cancer Cell Lines

doi: 10.3390/ijms23094654

Figure Lengend Snippet: Spectrophotometry results of VEGF B ELISA demonstrating the effects of PPV (10–300 µM) on proliferation on MDA-MB-231 cells compared to A549- and DU145 cell lines at 72 h. The average of three independent experiments is represented by the graph with error bars indicating standard deviation. The statistical significance of MDA-MB-231 cells is indicated with an asterisk (*), the statistical significance of A549 cells is indicated with two asterisks (**), and the statistical significance of DU145 cells is indicated with three asterisks (***). Statistical significance is represented by an * when using the student t -test with a p value of 0.05 compared to cells propagated in complete growth medium.

Article Snippet: The effects of PPV on the expression of VEGF ligand B and receptors 1 and 2 were investigated in MDA-MB-231, A549, and DU145 cells using a VEGF B ELISA kit (Elabscience biotechnology incorporated (Houston, TX, USA)) a VEGF R1 ELISA kit and a VEGF R2 ELISA kit (Abcam plc. (Cambridge, UK).

Techniques: Spectrophotometry, Enzyme-linked Immunosorbent Assay, Standard Deviation

Journal: International Journal of Molecular Sciences

Article Title: In Vitro Effects of Papaverine on Cell Migration and Vascular Endothelial Growth Factor in Cancer Cell Lines

doi: 10.3390/ijms23094654

Figure Lengend Snippet: Spectrophotometry results of VEGF R1 ELISA demonstrating the effects of PPV (10–300 µM) on proliferation on MDA-MB-231 cells compared to A549- and DU145 cell lines at 48 h. The average of three independent experiments is represented by the graph with error bars indicating standard deviation. The statistical significance of MDA-MB-231 cells is indicated with an asterisk (*), the statistical significance of A549 cells is indicated with two asterisks (**), and the statistical significance of DU145 cells is indicated with three asterisks (***). Statistical significance is represented by an * when using the student t -test with a P value of 0.05 compared to cells propagated in complete growth medium.

Article Snippet: The effects of PPV on the expression of VEGF ligand B and receptors 1 and 2 were investigated in MDA-MB-231, A549, and DU145 cells using a VEGF B ELISA kit (Elabscience biotechnology incorporated (Houston, TX, USA)) a VEGF R1 ELISA kit and a VEGF R2 ELISA kit (Abcam plc. (Cambridge, UK).

Techniques: Spectrophotometry, Enzyme-linked Immunosorbent Assay, Standard Deviation

Journal: International Journal of Molecular Sciences

Article Title: In Vitro Effects of Papaverine on Cell Migration and Vascular Endothelial Growth Factor in Cancer Cell Lines

doi: 10.3390/ijms23094654

Figure Lengend Snippet: Spectrophotometry results of VEGF R2 ELISA demonstrating the effects of PPV (10–300 µM) on proliferation on MDA-MB-231 cells compared to A549- and DU145 cell lines at 48 h. The average of three independent experiments is represented by the graph with error bars indicating standard deviation. The statistical significance of MDA-MB-231 cells is indicated with an asterisk (*), the statistical significance of A549 cells is indicated with two asterisks (**), and the statistical significance of DU145 cells is indicated with three asterisks (***). Statistical significance is represented by an * when using the student t -test with a p value of 0.05 compared to cells propagated in complete growth medium.

Article Snippet: The effects of PPV on the expression of VEGF ligand B and receptors 1 and 2 were investigated in MDA-MB-231, A549, and DU145 cells using a VEGF B ELISA kit (Elabscience biotechnology incorporated (Houston, TX, USA)) a VEGF R1 ELISA kit and a VEGF R2 ELISA kit (Abcam plc. (Cambridge, UK).

Techniques: Spectrophotometry, Enzyme-linked Immunosorbent Assay, Standard Deviation

Journal: International Journal of Molecular Sciences

Article Title: In Vitro Effects of Papaverine on Cell Migration and Vascular Endothelial Growth Factor in Cancer Cell Lines

doi: 10.3390/ijms23094654

Figure Lengend Snippet: Spectrophotometry results of FAK ELISA demonstrating the effects of PPV (10–300 µM) on proliferation on MDA-MB-231 cells compared to A549- and DU145 cell lines at 48 h. The average of there independent experiments is represented by the graph with error bars indicating standard deviation. The statistical significance of MDA-MB-231 cells is indicated with an asterisk (*), the statistical significance of A549 cells is indicated with two asterisks (**), and the statistical significance of DU145 cells is indicated with three asterisks (***). Statistical significance is represented by an * when using the student t -test with a p value of 0.05 compared to cells propagated in complete growth medium.

Article Snippet: The effects of PPV on the expression of VEGF ligand B and receptors 1 and 2 were investigated in MDA-MB-231, A549, and DU145 cells using a VEGF B ELISA kit (Elabscience biotechnology incorporated (Houston, TX, USA)) a VEGF R1 ELISA kit and a VEGF R2 ELISA kit (Abcam plc. (Cambridge, UK).

Techniques: Spectrophotometry, Enzyme-linked Immunosorbent Assay, Standard Deviation

Journal: Langmuir

Article Title: Profiling High-Abundance Serum Proteins in the Corona of Nanodiamonds Using Mass Spectrometry

doi: 10.1021/acs.langmuir.5c06674

Figure Lengend Snippet: PC influences the cytotoxicity of NDs. (a) Bright field images of A549 cells before and after exposure to oxDND and HPHT ND, with and without PC. (b) Survival rate of A549 cells before and after exposure to NDs, with and without PC, measured using the CCK-8 assay ( n = 3; * p < 0.05).

Article Snippet: The viability of A549 cells exposed to NDs, with and without PC, was determined using the CCK-8 assay (Elabscience, Japan).

Techniques: CCK-8 Assay

Journal: Viruses

Article Title: Mitochondrial Antiviral Signaling (MAVS) Protein Modulates the Transition from Acute to Persistent Parainfluenza Virus Infection and Resistance to Complement-Mediated Cell Lysis

doi: 10.3390/v18040416

Figure Lengend Snippet: Loss of MAVS impairs antiviral gene induction independent of STAT1. ( A ) WT or MAVS KO A549 cells were mock-infected or infected with Le-PIV5 at an MOI of 10 and harvested at 24 or 48 hpi. Total RNA was analyzed by quantitative PCR to assess expression of IFN-β, IFIT1, and OAS2. Gene expression levels are expressed relative to the corresponding mock-infected controls. ( B ) Protein lysates from WT or MAVS KO cells that were mock-infected or infected with Le-PIV5 at a MOI of 10 were collected at 24 and 48 hpi and analyzed by Western blotting for STAT1 protein. β-actin was used as a loading control. ** indicates a p value < 0.01, *** indicates a p value < 0.001, and **** indicates a p value < 0.0001.

Article Snippet: A549 KO-MAVS cells were purchased commercially (A549-DualTM KO-MAVS cells, catalog #a549d-komavs; InvivoGen, San Diego, CA, USA) and grown in DMEM 10% HI FBS, 100 U/mL penicillin, 100 μg/mL streptomycin, 100 μg/mL Normocin.

Techniques: Infection, Real-time Polymerase Chain Reaction, Expressing, Gene Expression, Western Blot, Control

Journal: Prostaglandins & other lipid mediators

Article Title: Diosgenin suppresses COX-2 and mPGES-1 via GR and improves LPS-induced liver injury in mouse.

doi: 10.1016/j.prostaglandins.2021.106580

Figure Lengend Snippet: Fig. 1. Suppression of cyclooxygenase (COX)-2 and microsomai prostaglandin E synthase-1 (mPGES-1) by diosgenin and recovery of COX-2 expression by glucocorticoid receptor (GR) antagonist via nuclear factor-kappa B (NF-κB) translocation. A: A549 cells were incubated with or without diosgenin (100 or 1,000 nM) or dexamethasone (Dex; 100 nM) for 48 h. The protein expression levels of COX-2 and mPGES-1 were analyzed by western blotting. The ratio was represented as a relative value against the control cells. B: AA549 cells were incubated with diosgenin (100 nM) for 24 h with a GR antagonist, RU486 (0–100 nM). The expression levels of PTGS2 (COX-2) were analyzed by quantitative RT-PCR and are shown as relative values against control cells without diosgenin, Dex, and RU486. Data are represented as the means ± S.E. of three separate experiments; p < 0.01 compared with *diosgenin (−)/Dex (−)/RU486 (−). C: Intracellular localization of NF-κB (red) and DAPI (blue) was visualized by CLSM. Scale bar: 20 μm.

Article Snippet: A549 cell lysates (6.6 × 105 cells) after the treatment, with or without diosgenin for 48 h, were subjected to western blotting using rabbit anti-COX-2 antibody (1:1,000, Novus Biochemicals LLC, Centennial, Co), rabbit anti-mPGES-1 antibody (1:250, Santa Cruz Biotechnology, Dallas, TX), or rabbit anti-β-actin antibody (1:1,000, Cell Signaling Technology, Boston, MA).

Techniques: Expressing, Translocation Assay, Incubation, Western Blot, Control, Quantitative RT-PCR

Journal: Nucleic Acids Research

Article Title: Inhibition of ATR protein kinase activity by schisandrin B in DNA damage response

doi: 10.1093/nar/gkp593

Figure Lengend Snippet: SchB treatment causes a decrease in cell viability after UV irradiation. Relative cell viability was measured by clonogenic assay. A549 cells were seeded as 500 cells/60-mm dish, and then incubated for 24 h before the experiment. The cells were subjected to 1 h pre-incubation and 14 days post-incubation ( A ) with SchB (0, 1, 10 and 30 μM) after ( B ) UV irradiation (0, 20 and 50 J/m 2 ) or ( C ) IR irradiation (0, 2 and 6 Gy). Values are expressed as mean ± SD ( n = 3). * P < 0.05 versus SchB untreated control.

Article Snippet: ATM was purified by immunoprecipitation from A549 cell lysates, using anti-ATM monoclonal antibody (Rockland Immunochemicals).

Techniques: Irradiation, Clonogenic Assay, Incubation, Control

Journal: Nucleic Acids Research

Article Title: Inhibition of ATR protein kinase activity by schisandrin B in DNA damage response

doi: 10.1093/nar/gkp593

Figure Lengend Snippet: SchB treatment abolishes cell cycle checkpoints induced by UV irradiation. ( A ) Effect of SchB on the G2/M checkpoint after UV-induced DNA damage using G2/M synchronized cells. Cell-cycle distributions were analyzed by FACS after 1 h of UV exposure, followed by data analysis using ModFit software. The histogram in gray represents cells synchronized at the G2/M phase by nocodazole. A549 cells were treated with 30 μM SchB or 10 mM caffeine 1 h before UV irradiation (0, 20 and 50 J/m 2 ). The arrow shows the cells distributed in the G1 phase. ( B ) Data were expressed as the percentage of G1 cells in relation to the total number of cells. ( C ) The percentage of mitotic cells was estimated by phosphorylation of histone H3 at Ser10. ( D ) The mitotic percentage of asynchronous cells was estimated by phosphorylation of histone H3 in 10 J/m 2 of UV irradiation followed by 1h incubation. ( E ) The percentage of DNA synthesizing cells was visualized by BrdU intake of cells. ( F ) Data obtained in E were analyzed using XL2 software to determine the percentage of mitotic cells. Values are expressed as mean ± SD ( n = 3). * P < 0.05 versus SchB untreated control.

Article Snippet: ATM was purified by immunoprecipitation from A549 cell lysates, using anti-ATM monoclonal antibody (Rockland Immunochemicals).

Techniques: Irradiation, Software, Phospho-proteomics, Incubation, Control

Journal: Nucleic Acids Research

Article Title: Inhibition of ATR protein kinase activity by schisandrin B in DNA damage response

doi: 10.1093/nar/gkp593

Figure Lengend Snippet: Inhibitory effects of SchB treatment on checkpoint proteins. ( A ) A549 cells were pre-incubated with or without 30 μM SchB for 1 h prior to induction of DNA damage. After pre-incubation, cells were subjected to 10 Gy of IR or 20 J/m 2 of UV irradiation, and cultured at 37°C for 1 h and 3 h, respectively. ( B ) Dose-dependent effects of SchB on the phosphorylation level of p53 and Chk1 were observed by adding increasing concentrations of SchB before and after induction of DNA damage by UV irradiation (20 J/m 2 ). A549 cells were incubated with a proteasome inhibitor (50 μM LLnL) throughout the experiments in A, B and C to avoid proteasome-dependent protein degradation. Equal loading of extracted proteins was confirmed by determining immuno-stained tubulin. ( C ) A549 cells were pre-incubated with or without 30 μM SchB or 10 mM caffeine for 1 h prior to induction of DNA damage. After pre-incubation, cells were subjected to UV irradiation (20 J/m 2 ), and cultured at 37°C for 3 h. ( D ) Flag-tagged ATR was expressed in HEK293T cells following transient transfection with a flag-tagged ATR, and the expressed proteins were immunoprecipitated. The association of ATR with ATRIP was analyzed by immunoblot analysis.

Article Snippet: ATM was purified by immunoprecipitation from A549 cell lysates, using anti-ATM monoclonal antibody (Rockland Immunochemicals).

Techniques: Incubation, Irradiation, Cell Culture, Phospho-proteomics, Staining, Transfection, Immunoprecipitation, Western Blot

Journal: Nucleic Acids Research

Article Title: Inhibition of ATR protein kinase activity by schisandrin B in DNA damage response

doi: 10.1093/nar/gkp593

Figure Lengend Snippet: SchB treatment inhibits ATR kinase activity. Kinase activities of ATR ( A ), ATM ( B ), Chk1 ( C ), PI3K ( D ), DNA-PK ( E ) and mTOR ( F ) were measured in vitro as phosphorylation activity in the presence of SchB. A flag-tagged ATR-wt plasmid was transfected into HEK293T cells. ATR protein kinases were purified by immunoprecipitation using anti-Flag M2 antibody and Protein-G agarose. The endogeneous ATM protein was purified from A549 cell lysates with anti-ATM antibody. Kinase activity was monitored for 20 min at 30°C. Chk1 (Upstate), PI3K (Echelon Biosciences), DNA-PK (Promega) and mTOR (Calbiochem) activities were measured using respective assay kits according to the manufacturer ’ s instructions. Values are presented as mean ± SD ( n = 3).

Article Snippet: ATM was purified by immunoprecipitation from A549 cell lysates, using anti-ATM monoclonal antibody (Rockland Immunochemicals).

Techniques: Activity Assay, In Vitro, Phospho-proteomics, Plasmid Preparation, Transfection, Purification, Immunoprecipitation

Journal: Nucleic Acids Research

Article Title: Inhibition of ATR protein kinase activity by schisandrin B in DNA damage response

doi: 10.1093/nar/gkp593

Figure Lengend Snippet: SchB prevents ATR-dependent signaling pathway after DNA damage. ( A ) AT2KY fibroblasts were pre-incubated with or without 30 μM SchB for 1 h, and then subjected to UV irradiation (20 J/m 2 ). The cells were harvested 4 h later and phosphorylation levels of p53, Chk1, SMC1 and BRCA1 were measured to assess checkpoint function. ( B ) A549 cells were transfected with siRNA for control (siGFP), ATM or ATR using the oligofectamine method, and the cell extracts were immunoblotted with anti-phosphorylated p53, phosphorylated Chk1, total-ATM, total-ATR or anti-tubulin. After 72 h incubation for the knockdown of ATM or ATR, cells were subjected to UV irradiation (20 J/m 2 ) followed by 4 h incubation with or without SchB (30 μM) pre/post-incubation. The equal loading of an extracted protein was confirmed using anti-monoclonal tubulin antibody. ( C ) The cell viability was measured in ATR-deficient Seckel syndrome fibroblasts with or without SchB (0, 1, 10 and 30 μM) after UV irradiation (0, 25, 50 and 75 J/m 2 ). Values are expressed as mean ± SD ( n = 3).

Article Snippet: ATM was purified by immunoprecipitation from A549 cell lysates, using anti-ATM monoclonal antibody (Rockland Immunochemicals).

Techniques: Incubation, Irradiation, Phospho-proteomics, Transfection, Control, Knockdown

Journal: Nucleic Acids Research

Article Title: Inhibition of ATR protein kinase activity by schisandrin B in DNA damage response

doi: 10.1093/nar/gkp593

Figure Lengend Snippet: Phosphorylation of ERK at Thr202 and Tyr204. A549 cells were pre-treated with 50 μM PD098059 or 30 μM SchB after serum starvation for 16 h. The phosphorylation of ERK was activated by adding 100 ng/ml PMA followed by a 5 min incubation. The cells were lysed and applied to immunoblot analysis. Equal loading of an extracted protein was confirmed using anti-monoclonal tubulin antibody.

Article Snippet: ATM was purified by immunoprecipitation from A549 cell lysates, using anti-ATM monoclonal antibody (Rockland Immunochemicals).

Techniques: Phospho-proteomics, Incubation, Western Blot