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

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Detection of experimental and clinical immune complexes by measuring SHIP-1 recruitment to the inhibitory FcγRIIB

doi: 10.4049/jimmunol.1700832

Figure Lengend Snippet: (A-C) HEK293F cells, transiently co-transfected with CD32B-SmBiT, SHIP-1-LgBiT (N-terminus) alone (A), with CD32A (B) or ITIM-mutated CD32B-SmBiT, SHIP-1-LgBiT and CD32A (C) on d0, were stimulated with medium, IC or monomeric IgG (10µg/ml) on d1. Luminescence was measured at time 0 following substrate addition, and at 5-minute intervals. (D) HEK293F cells, transfected as in (B) were pre-treated with the Src family kinase inhibitor PP2 at the indicated concentrations, or DMSO control, on d1 following transfection, followed by stimulation with medium, IC or monomeric IgG (10µg/ml). Luminescence was measured as above. (A-D) Left - schematic diagrams; right - fold inductions, with means ± S.D. of technical replicates, representative of 3 (A), at least 3 (B) and 3 (C) independent experiments. (D) No responses to medium control or monomeric IgG were detected (data not shown). The absence of response to IC with prior 10µM PP2 treatment is representative of 3 independent experiments.

Article Snippet: The Src family kinase inhibitors PP2 and Dasatinib Monohydrate were from Selleckchem.

Techniques: Transfection

Journal: PLOS Genetics

Article Title: Fibroblast mechanoperception instructs pulmonary developmental and pattern specification gene expression programs

doi: 10.1371/journal.pgen.1011924

Figure Lengend Snippet: ( A ) Western blots for HOXA5 in WT and Thy-1 KO iHLFs cultured on 5kPa PDMS hydrogel or TCP following stimulation with 20μM SRC specific inhibitor KB SRC 4 or 10μM pan-SFK inhibitor PP2 for 6h. Quantification of HOXA5 normalized to loading control was plotted as a ratio of normalized signal relative to DMSO vehicle control. Mean ± S.D. plotted; N = 3 (Thy-1 KO -5kPa and WT-1GPa) or N = 4 (WT-5kPa and Thy-1 KO -1GPa) independent experiments; WT-5kPa and WT-1GPa = 1-way ANOVA with post-hoc uncorrected Fisher’s LSD test, Thy-1 KO -5kPa and Thy-1 KO -1GPa = non-parametric Kruskal-Wallis test with post-hoc uncorrected Dunn’s test. Tests chosen based on data distribution and variance. ( B ) Western blots for HOXA5 in WT and Thy-1 KO iHLFs cultured on 5kPa PDMS hydrogel or TCP substrates coated with αV-promoting (Fn 4G ) or α5-promoting (Fn 9*10 ) peptide fragments of the 9–10FnIII integrin binding site. Quantification of HOXA5 normalized to loading control was plotted as a ratio of normalized signal relative to full length human Fn (Fn Full ) control. Mean ± S.D. plotted; N = 3 (WT-5kPa, WT-1GPa, and Thy-1 KO -5kPa) or N = 4 (Thy-1 KO -1GPa) independent experiments; WT-5kPa = 1-way ANOVA with post-hoc uncorrected Fisher’s LSD test, Thy-1 KO -5kPa = Brown-Forsythe and Welch ANOVA with post-hoc unpaired t-tests with Welch’s correction, WT-1GPa and Thy-1 KO -1GPa = non-parametric Kruskal-Wallis test with post-hoc uncorrected Dunn’s test. Tests chosen based on data distribution and variance. For all statistical tests: ns = p > 0.05; p < 0.05 (*); p < 0.01 (**); p < .001 (***); p < .0001 (****).

Article Snippet: After allowing for adhesion, cells were stimulated with 1% FBS DMEM containing 20μM SRC inhibitor KB SRC 4 (Tocris) or 10μM pan-Src Family Kinase (SFK) inhibitor PP2 (Tocris) for periods of 3h or 6h.

Techniques: Western Blot, Cell Culture, Control, Binding Assay