Journal: PLoS ONE

Article Title: IgE by Itself Affects Mature Rat Mast Cell Preformed and De Novo -Synthesized Mediator Release and Amplifies Mast Cell Migratory Response

doi: 10.1371/journal.pone.0079286

Figure Lengend Snippet: Inhibition of IgE-induced mast cell histamine release, cysLT generation and TNF synthesis by various inhibitors.

Article Snippet: NaCl, KCl, MgCl 2 , CaCl 2 , 2-hydroxyethylpiperazine-N'-ethanesulphonic acid (HEPES), NaOH, glucose, HCl, o -phthaldehyde (OPT), Percoll, hematoxylin, toluidine blue, trypan blue, bovine serum albumin (BSA), PLC/PLA 2 inhibitor (U73122), PI3K inhibitor (LY294002), Src kinase inhibitor (Src Inhibitor-1, Src I-1), RIPA buffer and laminin from human placenta were obtained from Sigma-Aldrich (St. Louis, MO, USA).

Techniques: Inhibition

Journal: PLoS ONE

Article Title: IgE by Itself Affects Mature Rat Mast Cell Preformed and De Novo -Synthesized Mediator Release and Amplifies Mast Cell Migratory Response

doi: 10.1371/journal.pone.0079286

Figure Lengend Snippet: Mast cells were preincubated with U73122 (0.1 µM), Src I-1 (0.01 µM), LY294002 (50 µM) or medium alone for 1 h (none). Then, mast cells were treated with IgE at 5 µg/mL, washed and spontaneous (A), CCL5-induced (B) and TNF-induced (C) migration was examined in a Boyden microchamber. Bars for the positive controls demonstrate the same data set as in . Results are presented as the mean ± SEM of four independent experiments and each experiment was carried out in duplicate (n = 4). *p<0.05, **p<0.01, ***p<0.001.

Article Snippet: NaCl, KCl, MgCl 2 , CaCl 2 , 2-hydroxyethylpiperazine-N'-ethanesulphonic acid (HEPES), NaOH, glucose, HCl, o -phthaldehyde (OPT), Percoll, hematoxylin, toluidine blue, trypan blue, bovine serum albumin (BSA), PLC/PLA 2 inhibitor (U73122), PI3K inhibitor (LY294002), Src kinase inhibitor (Src Inhibitor-1, Src I-1), RIPA buffer and laminin from human placenta were obtained from Sigma-Aldrich (St. Louis, MO, USA).

Techniques: Migration

Journal: Antioxidants

Article Title: Piperine Attenuates Lithocholic Acid-Stimulated Interleukin-8 by Suppressing Src/EGFR and Reactive Oxygen Species in Human Colorectal Cancer Cells

doi: 10.3390/antiox11030530

Figure Lengend Snippet: Piperine Inhibits LCA-Induced IL-8 Expression by Suppressing the Activation of ERK1/2, Src, AKT, and EGFR Signaling Pathways. ( A ) HCT-116 cells were pretreated with 30 μM concentration of SB-203580 (SB), 30 μM concentration of PD-98059 (PD), and 30 μM concentration of JNKi for 1 h, and incubated with 30 μM concentration of LCA for 4 h. Subsequently, the IL-8 mRNA level was measured through RT-qPCR. ( B ) HCT-116 cells pretreated with 30 μM SB, 30 μM PD, and 30 μM JNKi for 1 h were incubated with 30 μM LCA for 24 h followed by ELISA assay to determine the IL-8 expression level. ( C ) HCT-116 cells were transiently transfected with dominant-negative mutants of MEK-1 (K97 M), JNK (TAM67), or mutant p38 MAPK (mP38), and co-transfected with pGL2-IL-8. After incubation with 30 μM LCA for 4 h, the luciferase activity was measured using a luminometer. ( D ) HCT-116 cells pretreated with 30 μM SB, 30 μM PD, or 30 μM JNKi for 1 h were incubated with 30 μM LCA for 4 h, and cell lysates were analyzed for the phosphorylated and total ERK level using western blotting. ( E ) HCT-116 cells pretreated with 5 mM NAC or 10 μM DPI for 1 h were incubated with 30 μM LCA for 4 h, and cell lysates were analyzed for the phosphorylated and total ERK level using western blotting. ( F ) HCT-116 cells were incubated with 30 μM LCA for 0–60 min, and cell lysates were analyzed for levels of phosphorylated Src, AKT, and EGFR using western blotting. ( G ) HCT-116 cells pretreated with 10 μM AG1478 (AG), 10 μM PP1, 10 μM PP2, and 20 μM LY-294002 (LY) for 1 h were incubated with 30 μM LCA for 4 h. Subsequently, the IL-8 mRNA level was measured using RT-PCR. ( H ) HCT-116 cells pretreated with 10 μM AG, 10 μM PP1, 10 μM PP2, and 20 μM LY for 1 h were incubated with 30 μM LCA for 4 h, and the luciferase activity was measured using a luminometer. ( I ) Cells transfected with si-Con, si-Src, si-EGFR, and si-AKT were incubated with 30 μM LCA for 4 h, and IL-8 mRNA level was measured using RT-PCR. ( J ) Effects of si-Src, si-AKT, and si-EGFR on LCA-stimulated IL-8 promoter activity in CRC cells. Cells transfected with si-Con, si-Src, si-EGFR, and si-AKT were incubated with 30 μM LCA for 4 h, and the luciferase activity was measured using a luminometer. Data represent the mean ± SEM from three experimental trials. * p < 0.05 versus control; # p < 0.05 versus LCA only. ( K ) HCT-116 cells pretreated with piperine in a dose-dependent manner for 1 h were incubated with 30 μM LCA for 30 min, and cell lysates were analyzed for the phosphorylated Src, EGFR, and AKT levels using western blotting.

Article Snippet: The Src family kinase inhibitors (PP2), LY-294002 (LY), SB-203580 (SB), and the JNK inhibitor (JNKi), BAY-11-7082 (BAY), and SN50AG-1478 (AG) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Expressing, Activation Assay, Protein-Protein interactions, Concentration Assay, Incubation, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Transfection, Dominant Negative Mutation, Mutagenesis, Luciferase, Activity Assay, Western Blot, Reverse Transcription Polymerase Chain Reaction, Control

Journal: Antioxidants

Article Title: Piperine Attenuates Lithocholic Acid-Stimulated Interleukin-8 by Suppressing Src/EGFR and Reactive Oxygen Species in Human Colorectal Cancer Cells

doi: 10.3390/antiox11030530

Figure Lengend Snippet: Inhibitory Role of Piperine in Activation of NADPH Oxidase-Derived Reactive Oxygen Species (ROS) during LCA-Induced IL-8 Expression in CRC Cells. ( A ) HCT-116 cells pretreated with N -acetyl- l -cysteine (NAC), diphenyleneiodonium chloride (DPI), or piperine for 1 h were incubated with 30 μM LCA for 10 min. Cells were then treated with 5 μg/mL of 5- and 6-carboxyl 2′,7′-dichlorodihydro-fluorescein diacetate (DCFDA) in the dark for 10 min. DCF fluorescence was imaged using a confocal laser scanning fluorescence microscope, and the statistically significant values of ROS production are presented. Scale bar: 100 μm. ( B ) Cells pretreated with NAC or DPI for 1 h were incubated with 30 μM LCA for 4 h, followed by mRNA extraction and RT-PCR to determine IL-8 expression. ( C ) HCT-116 cells were transiently transfected with 500 ng pGL2-IL-8 promoter–reporter construct. These transfected cells were pretreated with NAC or DPI for 1 h and incubated with 30 μΜ LCA for 4 h, and the luciferase activity was measured using a luminometer. Data represent the mean ± SEM from three experimental trials. * p < 0.05 versus control; # p < 0.05 versus LCA. ( D ) HCT-116 cells transfected with si-Con or si-p47 phox were incubated with 30 μM LCA for 30 min; cell lysates were analyzed for p47 phox level using western blotting. ( E ) HCT-116 cells was transfected with si-Con or si-p47 phox were incubated with 30 μM LCA for 4 h, and IL-8 mRNA level was measured using RT-PCR. ( F ) The NADPH oxidase activity measured by piperine pretreatment and LCA treatment. Data represent the mean ± SEM from three experimental trials. * p < 0.05 versus control; # p < 0.05 versus LCA. ( G ) HCT-116 cells pretreated with AG, PP1, PP2, or piperine for 1 h were incubated with 30 μM LCA for 4 h, and cell lysates were analyzed for p47 phox level using western blot analysis.

Article Snippet: The Src family kinase inhibitors (PP2), LY-294002 (LY), SB-203580 (SB), and the JNK inhibitor (JNKi), BAY-11-7082 (BAY), and SN50AG-1478 (AG) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Activation Assay, Derivative Assay, Expressing, Incubation, Fluorescence, Microscopy, Extraction, Reverse Transcription Polymerase Chain Reaction, Transfection, Construct, Luciferase, Activity Assay, Control, Western Blot

Journal: Antioxidants

Article Title: Piperine Attenuates Lithocholic Acid-Stimulated Interleukin-8 by Suppressing Src/EGFR and Reactive Oxygen Species in Human Colorectal Cancer Cells

doi: 10.3390/antiox11030530

Figure Lengend Snippet: Effect of Inhibitors on Expression of EGFR, Src, ERK, and AKT in HCT-116 Cells. ( A ) HCT-116 cells pretreated with 10 μM AG1478 (AG), 10 μM PP2, 30 μM PD-98059 (PD), or 20 μM LY-294002 (LY) for 1 h were incubated with 30 μM LCA, and cell lysates were analyzed for the phosphorylation level of EGFR, Src, ERK1/2, and AKT using western blotting. ( B ) HCT-116 cells pretreated with PD for 1 h were incubated with 30 μM LCA, and cell lysates were analyzed for the phosphorylation level of c-Fos and c-Jun through western blot analysis. ( C ) HCT-116 cells pretreated with AG, PP2, PD, or LY for 1 h were treated with 30 μM LCA, and cell lysates were analyzed for the phosphorylation level of p65 using western blot analysis. HCT-116 cells were transiently transfected with AP-1 luciferase reporter construct ( D ) or NF-κB luciferase reporter construct ( E ). These transfected cells were pretreated with 2 μM SR-11302 (SR), 5 mM N -acetyl- l -cysteine (NAC), 30 μM PD, 20 μM LY, or 10 μM BAY-11-7082 (BAY) for 1 h and incubated with 30 μΜ LCA for 4 h, and the luciferase activity was measured using a luminometer. Data represent the mean ± SEM from three experimental trials. * p < 0.05 versus control; # p < 0.05 versus LCA.

Article Snippet: The Src family kinase inhibitors (PP2), LY-294002 (LY), SB-203580 (SB), and the JNK inhibitor (JNKi), BAY-11-7082 (BAY), and SN50AG-1478 (AG) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Expressing, Incubation, Phospho-proteomics, Western Blot, Transfection, Luciferase, Construct, Activity Assay, Control