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Biomol GmbH sb202190
Involvement of PKC α -dependent p38 MAPK and JNK1/2 in TNF- α -mediated ROS generation and cPLA 2 expression. (a) Cells were pretreated with U0126, <t>SB202190,</t> or SP600125, or combinatorial treatment for 1 h, and then incubated with TNF- α for 16 h. The expression of cPLA 2 was determined by Western blotting. (b) Cells were pretreated with or without U0126, SB202190, or SP600125 for 1 h and then incubated with TNF- α for the indicated time intervals. (c) Cells were transfected with scrambled, p38, or JNK1 siRNA and then incubated with TNF- α for 16 h. The levels of p38, JNK1, and cPLA 2 were determined by Western blotting. (d) Cells were pretreated with Gö6976 for 1 h and then stimulated with TNF- α for 30 min. The levels of phospho-p38 MAPK and phospho-JNK1/2 were determined by Western blotting. (e) Cells were pretreated with NAC for 1 h, and then incubated with TNF- α for the indicated time intervals. The levels of phospho-p38 MAPK and phospho-JNK1/2 were determined by Western blotting. (f) Cells were pretreated with SB202190 (1 μ M) or SP600125 (1 μ M) for 1 h before exposure to TNF- α for 1 h (shaded bars) or 2 h (open bars). The NOX activity (shaded bars) and ROS generation (open bars) were analyzed. (g) Cells were pretreated with SB202190 (1 μ M) or SP600125 (1 μ M) for 1 h before exposure to TNF- α for 1 h. The membrane (ME) and cytosolic (CE) fractions were prepared and subjected to Western blotting using an anti-p47 phox antibody. All analyses were performed on samples from 4 RA patients. Results are representative of 3 independent experiments. Values in (a), (c), (f), and (g) are the mean ± SEM. * P
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1) Product Images from "HO-1 Induction by CO-RM2 Attenuates TNF-α-Induced Cytosolic Phospholipase A2 Expression via Inhibition of PKCα-Dependent NADPH Oxidase/ROS and NF-κB"

Article Title: HO-1 Induction by CO-RM2 Attenuates TNF-α-Induced Cytosolic Phospholipase A2 Expression via Inhibition of PKCα-Dependent NADPH Oxidase/ROS and NF-κB

Journal: Mediators of Inflammation

doi: 10.1155/2014/279171

Involvement of PKC α -dependent p38 MAPK and JNK1/2 in TNF- α -mediated ROS generation and cPLA 2 expression. (a) Cells were pretreated with U0126, SB202190, or SP600125, or combinatorial treatment for 1 h, and then incubated with TNF- α for 16 h. The expression of cPLA 2 was determined by Western blotting. (b) Cells were pretreated with or without U0126, SB202190, or SP600125 for 1 h and then incubated with TNF- α for the indicated time intervals. (c) Cells were transfected with scrambled, p38, or JNK1 siRNA and then incubated with TNF- α for 16 h. The levels of p38, JNK1, and cPLA 2 were determined by Western blotting. (d) Cells were pretreated with Gö6976 for 1 h and then stimulated with TNF- α for 30 min. The levels of phospho-p38 MAPK and phospho-JNK1/2 were determined by Western blotting. (e) Cells were pretreated with NAC for 1 h, and then incubated with TNF- α for the indicated time intervals. The levels of phospho-p38 MAPK and phospho-JNK1/2 were determined by Western blotting. (f) Cells were pretreated with SB202190 (1 μ M) or SP600125 (1 μ M) for 1 h before exposure to TNF- α for 1 h (shaded bars) or 2 h (open bars). The NOX activity (shaded bars) and ROS generation (open bars) were analyzed. (g) Cells were pretreated with SB202190 (1 μ M) or SP600125 (1 μ M) for 1 h before exposure to TNF- α for 1 h. The membrane (ME) and cytosolic (CE) fractions were prepared and subjected to Western blotting using an anti-p47 phox antibody. All analyses were performed on samples from 4 RA patients. Results are representative of 3 independent experiments. Values in (a), (c), (f), and (g) are the mean ± SEM. * P
Figure Legend Snippet: Involvement of PKC α -dependent p38 MAPK and JNK1/2 in TNF- α -mediated ROS generation and cPLA 2 expression. (a) Cells were pretreated with U0126, SB202190, or SP600125, or combinatorial treatment for 1 h, and then incubated with TNF- α for 16 h. The expression of cPLA 2 was determined by Western blotting. (b) Cells were pretreated with or without U0126, SB202190, or SP600125 for 1 h and then incubated with TNF- α for the indicated time intervals. (c) Cells were transfected with scrambled, p38, or JNK1 siRNA and then incubated with TNF- α for 16 h. The levels of p38, JNK1, and cPLA 2 were determined by Western blotting. (d) Cells were pretreated with Gö6976 for 1 h and then stimulated with TNF- α for 30 min. The levels of phospho-p38 MAPK and phospho-JNK1/2 were determined by Western blotting. (e) Cells were pretreated with NAC for 1 h, and then incubated with TNF- α for the indicated time intervals. The levels of phospho-p38 MAPK and phospho-JNK1/2 were determined by Western blotting. (f) Cells were pretreated with SB202190 (1 μ M) or SP600125 (1 μ M) for 1 h before exposure to TNF- α for 1 h (shaded bars) or 2 h (open bars). The NOX activity (shaded bars) and ROS generation (open bars) were analyzed. (g) Cells were pretreated with SB202190 (1 μ M) or SP600125 (1 μ M) for 1 h before exposure to TNF- α for 1 h. The membrane (ME) and cytosolic (CE) fractions were prepared and subjected to Western blotting using an anti-p47 phox antibody. All analyses were performed on samples from 4 RA patients. Results are representative of 3 independent experiments. Values in (a), (c), (f), and (g) are the mean ± SEM. * P

Techniques Used: Expressing, Incubation, Western Blot, Transfection, Activity Assay

TNF- α -mediated ROS-dependent activation of NF- κ B is required for TNF- α -induced cPLA 2 expression. (a) RASFs were pretreated with helenalin for 1 h and then incubated with TNF- α for 16 h. The expression of cPLA 2 was determined by Western blotting. (b) Cells were transfected with scrambled or p65 siRNA and then incubated with TNF- α for 16 h. The levels of p65 and cPLA 2 protein were determined by Western blotting. (c) Cells were pretreated with or without Gö6976 (1 μ M), SB202190 (1 μ M), SP600125 (1 μ M), or NAC (10 mM) for 1 h before exposure to TNF- α for the indicated time intervals. The levels of phospho-IKK α / β and phospho-p65 were determined by Western blotting. Cells were pretreated with Gö6976 (1 μ M), SB201290 (1 μ M), SP600125 (1 μ M), NAC (10 mM), or helenalin (1 μ M) for 1 h and then incubated with TNF- α for 30 min. (d) Cells were fixed and then labeled using an anti-p65 antibody and FITC-conjugated secondary antibody. (e) The nuclear extract (NE) was prepared and subjected to Western blotting using the indicated antibodies and lamin A used as an internal control. (f) Cells were transiently transfected with NF- κ B-Luc reporter gene, pretreated with Gö6976, SB201290, SP600125, DPI, APO, NAC, or helenalin for 1 h, and then incubated with TNF- α for 4 h. The promoter activity was determined. (g) Cells were pretreated with Gö6976, SB201290, SP600125, DPI, or NAC for 1 h and incubated with TNF- α for 2 h. Chromatin was immunoprecipitated using an anti-p65 antibody. The associated cPLA 2 promoter DNA was amplified by polymerase chain reaction (PCR). Input represents PCR products from chromatin pellets before immunoprecipitation. (h) Cells were transfected without or with a cPLA 2 -Luc reporter gene, pretreated with Gö6976, SB201290, SP600125, DPI, APO, NAC, or helenalin for 1 h, and then incubated with TNF- α for 6 h. The mRNA levels of cPLA 2 (open bars) and cPLA 2 -luc promoter (shaded bars) were measured. (i) The PGE 2 levels in the media were analyzed using a PGE 2 EIA kit. All analyses were performed on samples from 4 RA patients. Results are representative of 3 independent experiments. Values in (a), (b), (e), (f), and (g) are the mean ± SEM. * P
Figure Legend Snippet: TNF- α -mediated ROS-dependent activation of NF- κ B is required for TNF- α -induced cPLA 2 expression. (a) RASFs were pretreated with helenalin for 1 h and then incubated with TNF- α for 16 h. The expression of cPLA 2 was determined by Western blotting. (b) Cells were transfected with scrambled or p65 siRNA and then incubated with TNF- α for 16 h. The levels of p65 and cPLA 2 protein were determined by Western blotting. (c) Cells were pretreated with or without Gö6976 (1 μ M), SB202190 (1 μ M), SP600125 (1 μ M), or NAC (10 mM) for 1 h before exposure to TNF- α for the indicated time intervals. The levels of phospho-IKK α / β and phospho-p65 were determined by Western blotting. Cells were pretreated with Gö6976 (1 μ M), SB201290 (1 μ M), SP600125 (1 μ M), NAC (10 mM), or helenalin (1 μ M) for 1 h and then incubated with TNF- α for 30 min. (d) Cells were fixed and then labeled using an anti-p65 antibody and FITC-conjugated secondary antibody. (e) The nuclear extract (NE) was prepared and subjected to Western blotting using the indicated antibodies and lamin A used as an internal control. (f) Cells were transiently transfected with NF- κ B-Luc reporter gene, pretreated with Gö6976, SB201290, SP600125, DPI, APO, NAC, or helenalin for 1 h, and then incubated with TNF- α for 4 h. The promoter activity was determined. (g) Cells were pretreated with Gö6976, SB201290, SP600125, DPI, or NAC for 1 h and incubated with TNF- α for 2 h. Chromatin was immunoprecipitated using an anti-p65 antibody. The associated cPLA 2 promoter DNA was amplified by polymerase chain reaction (PCR). Input represents PCR products from chromatin pellets before immunoprecipitation. (h) Cells were transfected without or with a cPLA 2 -Luc reporter gene, pretreated with Gö6976, SB201290, SP600125, DPI, APO, NAC, or helenalin for 1 h, and then incubated with TNF- α for 6 h. The mRNA levels of cPLA 2 (open bars) and cPLA 2 -luc promoter (shaded bars) were measured. (i) The PGE 2 levels in the media were analyzed using a PGE 2 EIA kit. All analyses were performed on samples from 4 RA patients. Results are representative of 3 independent experiments. Values in (a), (b), (e), (f), and (g) are the mean ± SEM. * P

Techniques Used: Activation Assay, Expressing, Incubation, Western Blot, Transfection, Labeling, Activity Assay, Immunoprecipitation, Amplification, Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay

2) Product Images from "IL-1? Promotes Corneal Epithelial Cell Migration by Increasing MMP-9 Expression through NF-?B- and AP-1-Dependent Pathways"

Article Title: IL-1? Promotes Corneal Epithelial Cell Migration by Increasing MMP-9 Expression through NF-?B- and AP-1-Dependent Pathways

Journal: PLoS ONE

doi: 10.1371/journal.pone.0057955

IL-1β stimulates AP-1 and NF-κB promoter activation leading to cell migration. (A) Cells were pretreated with U0126, SP600125, or Bay11-7082 for 1 h, and then treated with IL-1β for 15 min. The translocation of p65 was observed using a fluorescence microscope. (B) Cells were pretreated with Bay11-7082, U0126, or SP600125, and then incubated with IL-1β for 8 h. The NF-κB promoter activity was measured. (C) Cells were pretreated with Tanshinone IIA, U0126, or SP600125 for 1 h, and then incubated with IL-1β for 8 h. The AP-1 promoter activity was measured. (D) Cells were pretreated with Bay11-7082, Tanshinone IIA, SB202190, U0126, or SP600125 for 1 h, and then incubated with IL-1β for 24 h. SIRCs migration was observed. Data are expressed as mean±S.E.M. of three independent experiments. * P
Figure Legend Snippet: IL-1β stimulates AP-1 and NF-κB promoter activation leading to cell migration. (A) Cells were pretreated with U0126, SP600125, or Bay11-7082 for 1 h, and then treated with IL-1β for 15 min. The translocation of p65 was observed using a fluorescence microscope. (B) Cells were pretreated with Bay11-7082, U0126, or SP600125, and then incubated with IL-1β for 8 h. The NF-κB promoter activity was measured. (C) Cells were pretreated with Tanshinone IIA, U0126, or SP600125 for 1 h, and then incubated with IL-1β for 8 h. The AP-1 promoter activity was measured. (D) Cells were pretreated with Bay11-7082, Tanshinone IIA, SB202190, U0126, or SP600125 for 1 h, and then incubated with IL-1β for 24 h. SIRCs migration was observed. Data are expressed as mean±S.E.M. of three independent experiments. * P

Techniques Used: Activation Assay, Migration, Translocation Assay, Fluorescence, Microscopy, Incubation, Activity Assay

p38 MAPK activation is not involved in IL-1β-induced MMP-9 expression. (A) Cells were pretreated with SB202190 for 1 h, and then incubated with IL-1β for 12 h. The conditioned media were subjected to determine MMP-9 expression. (B, C) Cells were pretreated with SB202190, and then incubated with IL-1β for 4 h (mRNA levels) or 12 h (promoter activity). The mRNA levels and promoter activity of MMP-9 were determined. (D) Cells were treated with IL-1β for the indicated time intervals or pretreated with SB202190, and then treated with IL-1β for 30 min. The phospho-p38 MAPK protein expression was determined by Western blotting. Data are expressed as mean±S.E.M. of three independent experiments. * P
Figure Legend Snippet: p38 MAPK activation is not involved in IL-1β-induced MMP-9 expression. (A) Cells were pretreated with SB202190 for 1 h, and then incubated with IL-1β for 12 h. The conditioned media were subjected to determine MMP-9 expression. (B, C) Cells were pretreated with SB202190, and then incubated with IL-1β for 4 h (mRNA levels) or 12 h (promoter activity). The mRNA levels and promoter activity of MMP-9 were determined. (D) Cells were treated with IL-1β for the indicated time intervals or pretreated with SB202190, and then treated with IL-1β for 30 min. The phospho-p38 MAPK protein expression was determined by Western blotting. Data are expressed as mean±S.E.M. of three independent experiments. * P

Techniques Used: Activation Assay, Expressing, Incubation, Activity Assay, Western Blot

AP-1 is required for IL-1β-induced MMP-9 expression. (A) Cells were pretreated with Tanshinone IIA for 1 h, and then incubated with IL-1β for 12 h. The conditioned media were subjected to determine MMP-9 expression. (B, C) Cells were pretreated with Tanshinone IIA, and then incubated with IL-1β for 4 h (mRNA levels) or 12 h (promoter activity). The mRNA levels and promoter activity of MMP-9 were determined. (D) Cells were transfected with wild-type MMP-9 promoter and AP-1-mutated MMP-9 promoter, and then incubated with IL-1β for 12 h. The promoter activity of MMP-9 was determined. (E) Cells were treated with IL-1β for the indicated time intervals. The AP-1 promoter activity was measured. (F) Cells were treated with IL-1β for the indicated time intervals. The mRNA levels of c-Fos and c-Jun were determined. (G, H) Cells were treated with IL-1β for the indicated time intervals or pretreated with U0126, SP600125, or SB202190, and then treated with IL-1β for 60 min. The levels of c-Jun phosphorylation were analyzed by Western blotting. Data are expressed as mean±S.E.M. of three independent experiments. * P
Figure Legend Snippet: AP-1 is required for IL-1β-induced MMP-9 expression. (A) Cells were pretreated with Tanshinone IIA for 1 h, and then incubated with IL-1β for 12 h. The conditioned media were subjected to determine MMP-9 expression. (B, C) Cells were pretreated with Tanshinone IIA, and then incubated with IL-1β for 4 h (mRNA levels) or 12 h (promoter activity). The mRNA levels and promoter activity of MMP-9 were determined. (D) Cells were transfected with wild-type MMP-9 promoter and AP-1-mutated MMP-9 promoter, and then incubated with IL-1β for 12 h. The promoter activity of MMP-9 was determined. (E) Cells were treated with IL-1β for the indicated time intervals. The AP-1 promoter activity was measured. (F) Cells were treated with IL-1β for the indicated time intervals. The mRNA levels of c-Fos and c-Jun were determined. (G, H) Cells were treated with IL-1β for the indicated time intervals or pretreated with U0126, SP600125, or SB202190, and then treated with IL-1β for 60 min. The levels of c-Jun phosphorylation were analyzed by Western blotting. Data are expressed as mean±S.E.M. of three independent experiments. * P

Techniques Used: Expressing, Incubation, Activity Assay, Transfection, Western Blot

3) Product Images from "Leptin Promotes cPLA2 Gene Expression through Activation of the MAPK/NF-κB/p300 Cascade"

Article Title: Leptin Promotes cPLA2 Gene Expression through Activation of the MAPK/NF-κB/p300 Cascade

Journal: International Journal of Molecular Sciences

doi: 10.3390/ijms161126045

Effects of garcinol and p300 siRNA on leptin-stimulated A549 cells. Serum-starved A549 cells were pretreated with the OB-R antibody (2 µg/mL), U0126 (10 µM), SB202190 (10 µM), SP600125 (10 µM), Bay11-7082 (10 µM), or garcinol (1 µM) for 1 h. Other cells were transfected with 100 nM p300 siRNA or scrambled siRNA as described in the Experimental section. The cells were then incubated with 1 µg/mL of leptin for the indicated time intervals. At the end of the incubation, the cells were harvested and cell lysates were extracted. ( A , B ) Expression of cPLA 2 -α and p300; ( C ) phosphorylation of p65; and ( D ) acetylation of histone H4 were detected using Western blot with specific antibodies. Cell membranes were stripped and reprobed with the anti-GAPDH antibody as internal controls. The data are expressed as mean ± SEM of 5 independent experiments ( n = 5). p
Figure Legend Snippet: Effects of garcinol and p300 siRNA on leptin-stimulated A549 cells. Serum-starved A549 cells were pretreated with the OB-R antibody (2 µg/mL), U0126 (10 µM), SB202190 (10 µM), SP600125 (10 µM), Bay11-7082 (10 µM), or garcinol (1 µM) for 1 h. Other cells were transfected with 100 nM p300 siRNA or scrambled siRNA as described in the Experimental section. The cells were then incubated with 1 µg/mL of leptin for the indicated time intervals. At the end of the incubation, the cells were harvested and cell lysates were extracted. ( A , B ) Expression of cPLA 2 -α and p300; ( C ) phosphorylation of p65; and ( D ) acetylation of histone H4 were detected using Western blot with specific antibodies. Cell membranes were stripped and reprobed with the anti-GAPDH antibody as internal controls. The data are expressed as mean ± SEM of 5 independent experiments ( n = 5). p

Techniques Used: Transfection, Incubation, Expressing, Western Blot

Effects of Bay11-7082 on leptin-regulated cPLA 2 -α expression and p65 phosphorylation. Serum-starved A549 cells were pretreated with different concentrations of Bay11-7082, OB-R (2 µg/mL), U0126 (10 µM), SB202190 (10 µM), SP600125 (10 µM), or Bay11-7082 (10 µM) for 1 h. The cells were then stimulated by 1 µg/mL of leptin for the indicated time intervals. At the end of incubation, the cells were harvested and cell lysates were extracted. ( A ) Expression of cPLA 2 -α and ( B ) Ser276 phosphorylation of p65 were detected using Western blot with specific antibodies. The cell membranes were stripped and reprobed with the anti-GAPDH antibody as internal controls. The data are expressed as mean ± SEM of five independent experiments ( n = 5). p
Figure Legend Snippet: Effects of Bay11-7082 on leptin-regulated cPLA 2 -α expression and p65 phosphorylation. Serum-starved A549 cells were pretreated with different concentrations of Bay11-7082, OB-R (2 µg/mL), U0126 (10 µM), SB202190 (10 µM), SP600125 (10 µM), or Bay11-7082 (10 µM) for 1 h. The cells were then stimulated by 1 µg/mL of leptin for the indicated time intervals. At the end of incubation, the cells were harvested and cell lysates were extracted. ( A ) Expression of cPLA 2 -α and ( B ) Ser276 phosphorylation of p65 were detected using Western blot with specific antibodies. The cell membranes were stripped and reprobed with the anti-GAPDH antibody as internal controls. The data are expressed as mean ± SEM of five independent experiments ( n = 5). p

Techniques Used: Expressing, Incubation, Western Blot

Effects of various inhibitors on leptin-stimulated cPLA 2 -α mRNA expression. Serum-starved A549 cells were pretreated with OB-R (2 µg/mL), PD98059 (10 µM), U0126 (10 µM), SB202190 (10 µM), SP600125 (10 µM), Bay11-7082 (10 µM), or garcinol (1 µM) for 1 h. The cells were then incubated with 1 µg/mL of leptin for 6 h. At the end of incubation, mRNA were extracted and used as templates of cDNA. Expression of cPLA 2 -α mRNA was detected using RT-PCR. Data are expressed as mean ± SEM of five independent experiments ( n = 5). p
Figure Legend Snippet: Effects of various inhibitors on leptin-stimulated cPLA 2 -α mRNA expression. Serum-starved A549 cells were pretreated with OB-R (2 µg/mL), PD98059 (10 µM), U0126 (10 µM), SB202190 (10 µM), SP600125 (10 µM), Bay11-7082 (10 µM), or garcinol (1 µM) for 1 h. The cells were then incubated with 1 µg/mL of leptin for 6 h. At the end of incubation, mRNA were extracted and used as templates of cDNA. Expression of cPLA 2 -α mRNA was detected using RT-PCR. Data are expressed as mean ± SEM of five independent experiments ( n = 5). p

Techniques Used: Expressing, Incubation, Reverse Transcription Polymerase Chain Reaction

SB202190 attenuation of cPLA 2 -α expression and p38 MAPK phosphorylation. Serum-starved A549 cells were pretreated with different concentrations of SB202190 or the OB-R antibody for 1 h and then stimulated by 1 µg/mL of leptin for the indicated time intervals. At the end of incubation, the cells were harvested and cell lysates were extracted. ( A ) Expression of cPLA 2 -α and ( B , C ) phosphorylation of p38 MAPK were detected using Western blot with specific antibodies. Cell membranes were stripped and reprobed with the anti-GAPDH antibody as internal controls. The data are expressed as mean ± SEM of five independent experiments ( n = 5). p
Figure Legend Snippet: SB202190 attenuation of cPLA 2 -α expression and p38 MAPK phosphorylation. Serum-starved A549 cells were pretreated with different concentrations of SB202190 or the OB-R antibody for 1 h and then stimulated by 1 µg/mL of leptin for the indicated time intervals. At the end of incubation, the cells were harvested and cell lysates were extracted. ( A ) Expression of cPLA 2 -α and ( B , C ) phosphorylation of p38 MAPK were detected using Western blot with specific antibodies. Cell membranes were stripped and reprobed with the anti-GAPDH antibody as internal controls. The data are expressed as mean ± SEM of five independent experiments ( n = 5). p

Techniques Used: Expressing, Incubation, Western Blot

4) Product Images from "TNF-? induces matrix metalloproteinase-9-dependent soluble intercellular adhesion molecule-1 release via TRAF2-mediated MAPKs and NF-?B activation in osteoblast-like MC3T3-E1 cells"

Article Title: TNF-? induces matrix metalloproteinase-9-dependent soluble intercellular adhesion molecule-1 release via TRAF2-mediated MAPKs and NF-?B activation in osteoblast-like MC3T3-E1 cells

Journal: Journal of Biomedical Science

doi: 10.1186/1423-0127-21-12

TNF-α-induced MMP-9 expression is mediated through the NF-κB element in MMP-9 promoter leading to soluble ICAM-1 release. (A) Cells were transiently transfected with a wild-type MMP-9 promoter-luciferase reporter construct (WT-MMP9), and then incubated with TNF-α for the indicated time intervals. (B) The WT-MMP9 transfected cells were pretreated with anti-TNFR1 neutralizing antibody (TNFR-Ab, 10 μg/ml), PP1 (3 μM), U0126 (3 μM), SB202190 (3 μM), SP600125 (3 μM), and Bay11-7082 (10 μM) for 1 h and then incubated with 15 ng/ml TNF-α for 6 h. (C) Cells were transfected with WT-MMP9 or mt-κB-MMP9 for 24 h and then incubated with TNF-α (15 ng/ml) for 6 h. The cell lysates were collected and determined the luciferase activity. (D,E) Cells were pretreated with GM6001 or MMP2/9i for 1 h and then incubated with TNF-α (15 ng/ml) for 24 h. (D, upper panel) Conditioned media were collected and analyzed by gelatin zymography to determine the MMP-9 expression. The conditioned media were analyzed by trichloroacetic acid-protein precipitation and Western blot using an anti-sICAM-1 antibody. (E) The cell lysates were analyzed by Western blot to determine the expression of ICAM-1. (D, lower panel) Cells were pretreated with MMP-2/9i (10 μM), PP1 (10 μM), U0126 (10 μM), SB202190 (10 μM), SP600125 (10 μM), or Bay11-7082 (10 μM) for 1 h and then incubated with TNF-α (15 ng/ml) for the indicated time intervals. The levels of sICAM-1 were determined in conditioned media using an sICAM-1 ELISA kit. Data are expressed as mean±SEM of three independent experiments. # P
Figure Legend Snippet: TNF-α-induced MMP-9 expression is mediated through the NF-κB element in MMP-9 promoter leading to soluble ICAM-1 release. (A) Cells were transiently transfected with a wild-type MMP-9 promoter-luciferase reporter construct (WT-MMP9), and then incubated with TNF-α for the indicated time intervals. (B) The WT-MMP9 transfected cells were pretreated with anti-TNFR1 neutralizing antibody (TNFR-Ab, 10 μg/ml), PP1 (3 μM), U0126 (3 μM), SB202190 (3 μM), SP600125 (3 μM), and Bay11-7082 (10 μM) for 1 h and then incubated with 15 ng/ml TNF-α for 6 h. (C) Cells were transfected with WT-MMP9 or mt-κB-MMP9 for 24 h and then incubated with TNF-α (15 ng/ml) for 6 h. The cell lysates were collected and determined the luciferase activity. (D,E) Cells were pretreated with GM6001 or MMP2/9i for 1 h and then incubated with TNF-α (15 ng/ml) for 24 h. (D, upper panel) Conditioned media were collected and analyzed by gelatin zymography to determine the MMP-9 expression. The conditioned media were analyzed by trichloroacetic acid-protein precipitation and Western blot using an anti-sICAM-1 antibody. (E) The cell lysates were analyzed by Western blot to determine the expression of ICAM-1. (D, lower panel) Cells were pretreated with MMP-2/9i (10 μM), PP1 (10 μM), U0126 (10 μM), SB202190 (10 μM), SP600125 (10 μM), or Bay11-7082 (10 μM) for 1 h and then incubated with TNF-α (15 ng/ml) for the indicated time intervals. The levels of sICAM-1 were determined in conditioned media using an sICAM-1 ELISA kit. Data are expressed as mean±SEM of three independent experiments. # P

Techniques Used: Expressing, Transfection, Luciferase, Construct, Incubation, Activity Assay, Zymography, Western Blot, Enzyme-linked Immunosorbent Assay

TNF-α stimulates c-Src-dependent MAPKs and NF-κB-dependent cascades in MC3T3-E1 cells. MC3T3-E1 cells were pretreated with 3 μM PP1 (A) transfected with c-Src siRNA (B) or pretreated with (C) U0126 (3 μM), SB202190 (3 μM), or SP600125 (3 μM) for 1 h, and then stimulated with TNF-α (15 ng/ml) for the indicated time intervals. The cell lysates were analyzed by Western blot using an anti-phospho-ERK1/2, anti-phospho-p38 MAPK, anti-phospho-JNK1/2, anti-phospho-IKKα/β, anti-phospho-p65, or anti-GAPDH (as a control) antibody. (D) Cells were pretreated with U0126 (3 μM), SB202190 (3 μM), SP600125 (3 μM), or TNF-α receptor 1 neutralized antibody (TNFR nAb) for 1 h and then stimulated with TNF-α (15 ng/ml) for 15 min. The translocation of p65 NF-κB was observed by immunofluorescence staining. (E) Cells were transfected with NF-κB-Luc construct, pretreated with U0126 (3 μM), SB202190 (3 μM), or SP600125 (3 μM) for 1 h, and then incubated with TNF-α (15 ng/ml) for 4 h. The cell lysates were collected and determined NF-κB-Luc activity. Similar results were obtained in three independent experiments.
Figure Legend Snippet: TNF-α stimulates c-Src-dependent MAPKs and NF-κB-dependent cascades in MC3T3-E1 cells. MC3T3-E1 cells were pretreated with 3 μM PP1 (A) transfected with c-Src siRNA (B) or pretreated with (C) U0126 (3 μM), SB202190 (3 μM), or SP600125 (3 μM) for 1 h, and then stimulated with TNF-α (15 ng/ml) for the indicated time intervals. The cell lysates were analyzed by Western blot using an anti-phospho-ERK1/2, anti-phospho-p38 MAPK, anti-phospho-JNK1/2, anti-phospho-IKKα/β, anti-phospho-p65, or anti-GAPDH (as a control) antibody. (D) Cells were pretreated with U0126 (3 μM), SB202190 (3 μM), SP600125 (3 μM), or TNF-α receptor 1 neutralized antibody (TNFR nAb) for 1 h and then stimulated with TNF-α (15 ng/ml) for 15 min. The translocation of p65 NF-κB was observed by immunofluorescence staining. (E) Cells were transfected with NF-κB-Luc construct, pretreated with U0126 (3 μM), SB202190 (3 μM), or SP600125 (3 μM) for 1 h, and then incubated with TNF-α (15 ng/ml) for 4 h. The cell lysates were collected and determined NF-κB-Luc activity. Similar results were obtained in three independent experiments.

Techniques Used: Transfection, Western Blot, Translocation Assay, Immunofluorescence, Staining, Construct, Incubation, Activity Assay

Involvement of p38 MAPK phosphorylation in TNF-α-induced MMP-9 expression. (A) Cells were pretreated with SB202190 for 1 h and then incubated with TNF-α (15 ng/ml) for 24 h. (B) Cells were pretreated with SB202190 (30 μM) for 1 h and then incubated with TNF-α (15 ng/ml) for 6 h. The isolated RNA samples were analyzed for the levels of MMP-9 mRNA by real-time PCR. Data are expressed as mean±SEM of three independent experiments. * P
Figure Legend Snippet: Involvement of p38 MAPK phosphorylation in TNF-α-induced MMP-9 expression. (A) Cells were pretreated with SB202190 for 1 h and then incubated with TNF-α (15 ng/ml) for 24 h. (B) Cells were pretreated with SB202190 (30 μM) for 1 h and then incubated with TNF-α (15 ng/ml) for 6 h. The isolated RNA samples were analyzed for the levels of MMP-9 mRNA by real-time PCR. Data are expressed as mean±SEM of three independent experiments. * P

Techniques Used: Expressing, Incubation, Isolation, Real-time Polymerase Chain Reaction

5) Product Images from "Cooperation of TLR2 with MyD88, PI3K, and Rac1 in Lipoteichoic Acid-Induced cPLA2/COX-2-Dependent Airway Inflammatory Responses"

Article Title: Cooperation of TLR2 with MyD88, PI3K, and Rac1 in Lipoteichoic Acid-Induced cPLA2/COX-2-Dependent Airway Inflammatory Responses

Journal: The American Journal of Pathology

doi: 10.2353/ajpath.2010.090714

LTA and S. aureus induce leukocytes in BAL and cPLA 2 and COX-2 expression in mice via PI3K/Akt, MAPKs, and NF-κB. Mice were given one dose of LY294002, U0126, SB202190, SP600125, or helenalin (2 mg/kg) for 1 hour before LTA ( A ) or live S. aureus
Figure Legend Snippet: LTA and S. aureus induce leukocytes in BAL and cPLA 2 and COX-2 expression in mice via PI3K/Akt, MAPKs, and NF-κB. Mice were given one dose of LY294002, U0126, SB202190, SP600125, or helenalin (2 mg/kg) for 1 hour before LTA ( A ) or live S. aureus

Techniques Used: Expressing, Mouse Assay

6) Product Images from "TNF-? Induces Cytosolic Phospholipase A2 Expression in Human Lung Epithelial Cells via JNK1/2- and p38 MAPK-Dependent AP-1 Activation"

Article Title: TNF-? Induces Cytosolic Phospholipase A2 Expression in Human Lung Epithelial Cells via JNK1/2- and p38 MAPK-Dependent AP-1 Activation

Journal: PLoS ONE

doi: 10.1371/journal.pone.0072783

TNF-α stimulates p300/ATF2/c-Jun/c-Fos complex formation. (A) Cells were transfected with scrambled or ATF2 siRNA, and then incubated with TNF-α for 24 h. The protein levels of ATF2 and cPLA 2 were determined. (B) Cells were incubated with TNF-α for the indicated time intervals. The levels of c-Fos, c-Jun, phospho-c-Jun, and phospho-ATF2 were determined. (C) Cells were pretreated with PD98059, SB202190, or SP600125, and then incubated with TNF-α for 90 min or 15 min. The levels of phospho-ATF2 and phospho-c-Jun were determined. (D) Cells were incubated with TNF-α for the indicated time intervals. The cell lysates were subjected to immunoprecipitation using an anti-p300 antibody, and then the immunoprecipitates were analyzed by Western blot using an anti-c-Fos, anti-c-Jun, anti-ATF2, or anti-p300 antibody. (E) Cells were treated with TNF-α for the indicated time intervals, and then ChIP assay was performed. Chromatin was immunoprecipitated using an anti-p300, anti-ATF2, anti-c-Fos, or anti-c-Jun antibody. One percent of the precipitated chromatin was assayed to verify equal loading (Input). Data are expressed as mean±S.E.M. of three independent experiments. # P
Figure Legend Snippet: TNF-α stimulates p300/ATF2/c-Jun/c-Fos complex formation. (A) Cells were transfected with scrambled or ATF2 siRNA, and then incubated with TNF-α for 24 h. The protein levels of ATF2 and cPLA 2 were determined. (B) Cells were incubated with TNF-α for the indicated time intervals. The levels of c-Fos, c-Jun, phospho-c-Jun, and phospho-ATF2 were determined. (C) Cells were pretreated with PD98059, SB202190, or SP600125, and then incubated with TNF-α for 90 min or 15 min. The levels of phospho-ATF2 and phospho-c-Jun were determined. (D) Cells were incubated with TNF-α for the indicated time intervals. The cell lysates were subjected to immunoprecipitation using an anti-p300 antibody, and then the immunoprecipitates were analyzed by Western blot using an anti-c-Fos, anti-c-Jun, anti-ATF2, or anti-p300 antibody. (E) Cells were treated with TNF-α for the indicated time intervals, and then ChIP assay was performed. Chromatin was immunoprecipitated using an anti-p300, anti-ATF2, anti-c-Fos, or anti-c-Jun antibody. One percent of the precipitated chromatin was assayed to verify equal loading (Input). Data are expressed as mean±S.E.M. of three independent experiments. # P

Techniques Used: Transfection, Incubation, Immunoprecipitation, Western Blot, Chromatin Immunoprecipitation

TNF-α induces leukocyte accumulation in BAL and cPLA 2 mRNA expression in mice via MAPKs and AP-1. (A) Mice were i.p. given one dose of PD98059, SB202190, SP600125, or Tanshinone IIA (2 mg/kg) for 1 h before TNF-α treatment, and sacrificed after 24 h. Lung tissues were homogenized to extract mRNA. The levels of cPLA 2 mRNA were determined by real-time PCR. (B, C) Mice were i.p. given one dose of PD98059, SB202190, SP600125, Tanshinone IIA, AACOCF 3 , AH 6809, SC-19220, or GW627368X (2 mg/kg) for 1 h before TNF-α treatment, and sacrificed after 24 h. BAL fluid was acquired and leukocyte count was determined by a hemocytometer. (D) Schematic representation of the signaling pathways involved in the TNF-α-induced cPLA 2 expression in HPAEpiCs. TNF-α-induced cPLA 2 expression and PGE 2 release are mediated through p38 MAPK- and JNK1/2-dependent p300/c-Fos/c-Jun/ATF2 complex formation in HPAEpiCs.
Figure Legend Snippet: TNF-α induces leukocyte accumulation in BAL and cPLA 2 mRNA expression in mice via MAPKs and AP-1. (A) Mice were i.p. given one dose of PD98059, SB202190, SP600125, or Tanshinone IIA (2 mg/kg) for 1 h before TNF-α treatment, and sacrificed after 24 h. Lung tissues were homogenized to extract mRNA. The levels of cPLA 2 mRNA were determined by real-time PCR. (B, C) Mice were i.p. given one dose of PD98059, SB202190, SP600125, Tanshinone IIA, AACOCF 3 , AH 6809, SC-19220, or GW627368X (2 mg/kg) for 1 h before TNF-α treatment, and sacrificed after 24 h. BAL fluid was acquired and leukocyte count was determined by a hemocytometer. (D) Schematic representation of the signaling pathways involved in the TNF-α-induced cPLA 2 expression in HPAEpiCs. TNF-α-induced cPLA 2 expression and PGE 2 release are mediated through p38 MAPK- and JNK1/2-dependent p300/c-Fos/c-Jun/ATF2 complex formation in HPAEpiCs.

Techniques Used: Expressing, Mouse Assay, Real-time Polymerase Chain Reaction

AP-1 is involved in TNF-α-induced cPLA 2 expression. (A) Cells were pretreated with Tanshinone IIA (TSIIA) for 1 h, and then incubated with TNF-α for 24 h. The protein levels of cPLA 2 were determined by Western blot. (B) Cells were pretreated with Tanshinone IIA (TSIIA), and then incubated with TNF-α for 6 h. cPLA 2 mRNA levels and promoter activity were determined. (C) Cells were transfected with scrambled, c-Jun, or c-Fos siRNA, and then incubated with TNF-α for 24 h. The protein levels of c-Jun, c-Fos, and cPLA 2 were determined. (D) Cells were transfected with pGL3-empty, wild-type cPLA 2 promoter, or AP-1-mutated cPLA 2 promoter, and then incubated with TNF-α for 6 h. The promoter activity of cPLA 2 was determined in the cell lysates. (E) Cells were pretreated with PD98059 (10 µM), SB202190 (10 µM), SP600125 (10 µM), or Tanshinone IIA (TSIIA; 10 µM) for 1 h, and then incubated with TNF-α for 24 h. The media were collected and analyzed for PGE 2 release. Data are expressed as mean±S.E.M. of three independent experiments. # P
Figure Legend Snippet: AP-1 is involved in TNF-α-induced cPLA 2 expression. (A) Cells were pretreated with Tanshinone IIA (TSIIA) for 1 h, and then incubated with TNF-α for 24 h. The protein levels of cPLA 2 were determined by Western blot. (B) Cells were pretreated with Tanshinone IIA (TSIIA), and then incubated with TNF-α for 6 h. cPLA 2 mRNA levels and promoter activity were determined. (C) Cells were transfected with scrambled, c-Jun, or c-Fos siRNA, and then incubated with TNF-α for 24 h. The protein levels of c-Jun, c-Fos, and cPLA 2 were determined. (D) Cells were transfected with pGL3-empty, wild-type cPLA 2 promoter, or AP-1-mutated cPLA 2 promoter, and then incubated with TNF-α for 6 h. The promoter activity of cPLA 2 was determined in the cell lysates. (E) Cells were pretreated with PD98059 (10 µM), SB202190 (10 µM), SP600125 (10 µM), or Tanshinone IIA (TSIIA; 10 µM) for 1 h, and then incubated with TNF-α for 24 h. The media were collected and analyzed for PGE 2 release. Data are expressed as mean±S.E.M. of three independent experiments. # P

Techniques Used: Expressing, Incubation, Western Blot, Activity Assay, Transfection

p38 MAPK is involved in TNF-α-induced cPLA 2 expression. (A) Cells were pretreated with SB202190 for 1 h, and then incubated with TNF-α for 24 h. The protein levels of cPLA 2 were determined by Western blot. (B) Cells were pretreated with SB202190 (10 µM) for 1 h, and then incubated with TNF-α for 6 h. cPLA 2 mRNA levels and promoter activity were determined. (C) Cells were transfected with scrambled or p38 siRNA, and then incubated with TNF-α for 24 h. The protein levels of p38 and cPLA 2 were determined. (D) Cells were pretreated with or without SB202190 (10 µM) for 1 h, and then incubated with TNF-α for the indicated time intervals. The levels of phospho-p38 MAPK were determined. Data are expressed as mean±S.E.M. of three independent experiments. # P
Figure Legend Snippet: p38 MAPK is involved in TNF-α-induced cPLA 2 expression. (A) Cells were pretreated with SB202190 for 1 h, and then incubated with TNF-α for 24 h. The protein levels of cPLA 2 were determined by Western blot. (B) Cells were pretreated with SB202190 (10 µM) for 1 h, and then incubated with TNF-α for 6 h. cPLA 2 mRNA levels and promoter activity were determined. (C) Cells were transfected with scrambled or p38 siRNA, and then incubated with TNF-α for 24 h. The protein levels of p38 and cPLA 2 were determined. (D) Cells were pretreated with or without SB202190 (10 µM) for 1 h, and then incubated with TNF-α for the indicated time intervals. The levels of phospho-p38 MAPK were determined. Data are expressed as mean±S.E.M. of three independent experiments. # P

Techniques Used: Expressing, Incubation, Western Blot, Activity Assay, Transfection

7) Product Images from "The CO donor CORM-2 inhibits LPS-induced vascular cell adhesion molecule-1 expression and leukocyte adhesion in human rheumatoid synovial fibroblasts"

Article Title: The CO donor CORM-2 inhibits LPS-induced vascular cell adhesion molecule-1 expression and leukocyte adhesion in human rheumatoid synovial fibroblasts

Journal: British Journal of Pharmacology

doi: 10.1111/bph.12680

Involvement of MAPKs, AP-1 and NF-κB in VCAM-1 promoter activity, mRNA expression, and leukocyte adhesion stimulated by LPS. (A) RASFs were transfected without or with VCAM-1 reporter gene, pretreated with 1 μM U0126 (U0), 10 μM SB202190 (SB), 1 μM SP600125 (SP), 1 μM helenalin (HLN) or 1 μM tanshinone IIA (TSIIA) for 1 h, and then incubated with LPS for 6 h (promoter) or 4 h (mRNA). Promoter activity and mRNA were determined. (B) Cells were pretreated with the indicated inhibitors for 1 h and then incubated with LPS for 8 h prior to addition of leukocytes. The leukocyte adherence was measured. Data are expressed as the mean and SEM of three separate experiments. In A and B, * P
Figure Legend Snippet: Involvement of MAPKs, AP-1 and NF-κB in VCAM-1 promoter activity, mRNA expression, and leukocyte adhesion stimulated by LPS. (A) RASFs were transfected without or with VCAM-1 reporter gene, pretreated with 1 μM U0126 (U0), 10 μM SB202190 (SB), 1 μM SP600125 (SP), 1 μM helenalin (HLN) or 1 μM tanshinone IIA (TSIIA) for 1 h, and then incubated with LPS for 6 h (promoter) or 4 h (mRNA). Promoter activity and mRNA were determined. (B) Cells were pretreated with the indicated inhibitors for 1 h and then incubated with LPS for 8 h prior to addition of leukocytes. The leukocyte adherence was measured. Data are expressed as the mean and SEM of three separate experiments. In A and B, * P

Techniques Used: Activity Assay, Expressing, Transfection, Incubation

LPS induces VCAM-1 expression via c-Src/MAPKs/AP-1. (A) RASFs were pretreated with tanshinone IIA or curcumin for 1 h and then incubated with LPS for 6 h. (B) Cells were transfected with an empty vector (scrb), c-Jun or c-Fos siRNA and then incubated with LPS for 6 h. The levels of VCAM-1 expression were analysed by Western blot. (C) Cells were treated with LPS (100 μg·mL −1 ) for the indicated time intervals. (D) RASFs were pretreated with the inhibitors and then incubated with LPS for 30 min. The levels of c-Jun and c-Fos mRNA were determined by real-time PCR. (E) Cells were transfected with AP-1 reporter gene together with a β-galactosidase plasmid, pretreated with the inhibitors for 1 h, and then incubated with LPS for 1 h. AP-1 promoter activity was determined. (F) Nuclear extracts were prepared from RASFs pretreated with U0126 (U0; 10 μM), SB202190 (SB; 10 μM), SP600125 (SP; 10 μM) or curcumin (Cur; 1 μM) and then incubated with LPS for 1 h. The complexes were subjected to EMSA followed by ECL detection. All analyses were performed on samples from three RA patients. Results are representative of three independent experiments. In A, C, * P ≤ 0.05; # P ≤ 0.01 versus vehicle alone. Values in B, D and E are the mean ± SEM. * P ≤ 0.05; # P ≤ 0.01 versus LPS alone.
Figure Legend Snippet: LPS induces VCAM-1 expression via c-Src/MAPKs/AP-1. (A) RASFs were pretreated with tanshinone IIA or curcumin for 1 h and then incubated with LPS for 6 h. (B) Cells were transfected with an empty vector (scrb), c-Jun or c-Fos siRNA and then incubated with LPS for 6 h. The levels of VCAM-1 expression were analysed by Western blot. (C) Cells were treated with LPS (100 μg·mL −1 ) for the indicated time intervals. (D) RASFs were pretreated with the inhibitors and then incubated with LPS for 30 min. The levels of c-Jun and c-Fos mRNA were determined by real-time PCR. (E) Cells were transfected with AP-1 reporter gene together with a β-galactosidase plasmid, pretreated with the inhibitors for 1 h, and then incubated with LPS for 1 h. AP-1 promoter activity was determined. (F) Nuclear extracts were prepared from RASFs pretreated with U0126 (U0; 10 μM), SB202190 (SB; 10 μM), SP600125 (SP; 10 μM) or curcumin (Cur; 1 μM) and then incubated with LPS for 1 h. The complexes were subjected to EMSA followed by ECL detection. All analyses were performed on samples from three RA patients. Results are representative of three independent experiments. In A, C, * P ≤ 0.05; # P ≤ 0.01 versus vehicle alone. Values in B, D and E are the mean ± SEM. * P ≤ 0.05; # P ≤ 0.01 versus LPS alone.

Techniques Used: Expressing, Incubation, Transfection, Plasmid Preparation, Western Blot, Real-time Polymerase Chain Reaction, Activity Assay

LPS induces VCAM-1 expression via c-Src-dependent MAPKs activation. (A) RASFs were pretreated with U0126, SB202190 or SP600125 for 1 h and then incubated with LPS for 6 h. (B) Cells were transfected with empty vector (pTOPO-U6) or with the indicated shRNA and then incubated with LPS for 6 h. (C) RASFs were pretreated with U0126 (U0; 10 μM), SB202190 (SB; 10 μM) or SP600125 (SP; 10 μM) and then incubated with LPS for the indicated time intervals. (D) Cells were transfected with an empty vector or c-Src shRNA and then incubated with LPS for the indicated time intervals. The levels of various proteins were analysed by Western blot using the antibodies indicated. All analyses were performed on samples from three RA patients. Results are representative of three independent experiments. Values (A–D) are the mean ± SEM. * P ≤ 0.05; # P ≤ 0.01 versus LPS alone.
Figure Legend Snippet: LPS induces VCAM-1 expression via c-Src-dependent MAPKs activation. (A) RASFs were pretreated with U0126, SB202190 or SP600125 for 1 h and then incubated with LPS for 6 h. (B) Cells were transfected with empty vector (pTOPO-U6) or with the indicated shRNA and then incubated with LPS for 6 h. (C) RASFs were pretreated with U0126 (U0; 10 μM), SB202190 (SB; 10 μM) or SP600125 (SP; 10 μM) and then incubated with LPS for the indicated time intervals. (D) Cells were transfected with an empty vector or c-Src shRNA and then incubated with LPS for the indicated time intervals. The levels of various proteins were analysed by Western blot using the antibodies indicated. All analyses were performed on samples from three RA patients. Results are representative of three independent experiments. Values (A–D) are the mean ± SEM. * P ≤ 0.05; # P ≤ 0.01 versus LPS alone.

Techniques Used: Expressing, Activation Assay, Incubation, Transfection, Plasmid Preparation, shRNA, Western Blot

8) Product Images from "PAR1-dependent COX-2/PGE2 production contributes to cell proliferation via EP2 receptors in primary human cardiomyocytes"

Article Title: PAR1-dependent COX-2/PGE2 production contributes to cell proliferation via EP2 receptors in primary human cardiomyocytes

Journal: British Journal of Pharmacology

doi: 10.1111/bph.12794

Effects of MAPKs in thrombin-induced COX-2 expression. (A) Cells were pretreated with SB202190, U0126, or SP600125 for 1 h and then incubated with thrombin for 16 h. The COX-2 protein expression was determined by Western blot. (B) Cells were pretreated
Figure Legend Snippet: Effects of MAPKs in thrombin-induced COX-2 expression. (A) Cells were pretreated with SB202190, U0126, or SP600125 for 1 h and then incubated with thrombin for 16 h. The COX-2 protein expression was determined by Western blot. (B) Cells were pretreated

Techniques Used: Expressing, Incubation, Western Blot

9) Product Images from "Transforming growth factor-?1 induces matrix metalloproteinase-9 and cell migration in astrocytes: roles of ROS-dependent ERK- and JNK-NF-?B pathways"

Article Title: Transforming growth factor-?1 induces matrix metalloproteinase-9 and cell migration in astrocytes: roles of ROS-dependent ERK- and JNK-NF-?B pathways

Journal: Journal of Neuroinflammation

doi: 10.1186/1742-2094-7-88

TGF-β1 induces MMP-9 expression and activation in the rat primary cultured astrocytes . (A) Time dependence of TGF-β1-induced MMP-9 expression and activation. Cells were treated with TGF-β1 (15 ng/ml) for the indicated time intervals. The conditioned media were collected and analyzed MMP-9 activity by gelatin zymography (upper panel). For MMP-9 protein level, conditioned media were immunoprecipitated with an anti-MMP-9 antibody and analyzed by western blot (lower panel). (B) Cells were pretreated with SB431542 (SB43, 10 μM), NAC (100 μM), U0126 (10 μM), SB202190 (SB20, 10 μM), SP600125 (SP, 10 μM), or Bay11-7082 (Bay, 1 μM) for 1 h, and then treated with TGF-β1 for 24 h. (C) Schematic pathway for TGF-β1-induced ROS-dependent MMP-9 expression and cell migration in RBA-1 cells. Each solid line and arrow represents a step in an activating pathway. TGF-β1 induces MMP-9 expression via TGF-β receptor, ROS-dependent activation of ERK1/2 and JNK1/2, and transcription factor NF-κB pathway, which results in the promotion of cell migration in RBA-1 cells.
Figure Legend Snippet: TGF-β1 induces MMP-9 expression and activation in the rat primary cultured astrocytes . (A) Time dependence of TGF-β1-induced MMP-9 expression and activation. Cells were treated with TGF-β1 (15 ng/ml) for the indicated time intervals. The conditioned media were collected and analyzed MMP-9 activity by gelatin zymography (upper panel). For MMP-9 protein level, conditioned media were immunoprecipitated with an anti-MMP-9 antibody and analyzed by western blot (lower panel). (B) Cells were pretreated with SB431542 (SB43, 10 μM), NAC (100 μM), U0126 (10 μM), SB202190 (SB20, 10 μM), SP600125 (SP, 10 μM), or Bay11-7082 (Bay, 1 μM) for 1 h, and then treated with TGF-β1 for 24 h. (C) Schematic pathway for TGF-β1-induced ROS-dependent MMP-9 expression and cell migration in RBA-1 cells. Each solid line and arrow represents a step in an activating pathway. TGF-β1 induces MMP-9 expression via TGF-β receptor, ROS-dependent activation of ERK1/2 and JNK1/2, and transcription factor NF-κB pathway, which results in the promotion of cell migration in RBA-1 cells.

Techniques Used: Expressing, Activation Assay, Cell Culture, Activity Assay, Zymography, Immunoprecipitation, Western Blot, Migration

TGF-β1-induced MMP-9 expression is mediated through JNK1/2, but not p38 MAPK . Cells were treated with TGF-β1 (15 ng/ml) for 16 h in the absence or presence of (A) SB202190 or (B) SP600125. (C) Cells were pretreated with SB202190 (SB, 10 μM) or SP600125 (SP, 10 μM) before exposure to TGF-β1 for 6 h. Total RNA was collected and analyzed by RT-PCR. (D) Time dependence of TGF-β1-stimulated JNK1/2 phosphorylation, cells were incubated with TGF-β1 (15 ng/ml) for the indicated times. Moreover, cells were treated with TGF-β1 for 4 h in the presence of SP600125 (SP/4, 10 μM). (E) Cells were transfected with an empty vector (pcDNA3, as a control) or dominant negative mutant of p38 MAPK (Δp38) or JNK (ΔJNK) for 24 h, and then exposed to TGF-β1 for 16 h. (F) For cell migration, cells were pretreated with U0126 (10 μM) or SP600125 (10 μM) for 1 h and then incubated with TGF-β1 (15 ng/ml) for 48 h. The image is representative of three similar experiments (n = 3). The conditioned media (A, B, E) were analyzed gelatin zymorgraphy, and the whole cell lysates (D) were analyzed using an anti-phospho-JNK1/2 or anti-GAPDH (as an internal control) antibody. Data are expressed as mean ± SEM (D) or mean (A, B, C, E) of at least three independent experiments (n = 3). * P
Figure Legend Snippet: TGF-β1-induced MMP-9 expression is mediated through JNK1/2, but not p38 MAPK . Cells were treated with TGF-β1 (15 ng/ml) for 16 h in the absence or presence of (A) SB202190 or (B) SP600125. (C) Cells were pretreated with SB202190 (SB, 10 μM) or SP600125 (SP, 10 μM) before exposure to TGF-β1 for 6 h. Total RNA was collected and analyzed by RT-PCR. (D) Time dependence of TGF-β1-stimulated JNK1/2 phosphorylation, cells were incubated with TGF-β1 (15 ng/ml) for the indicated times. Moreover, cells were treated with TGF-β1 for 4 h in the presence of SP600125 (SP/4, 10 μM). (E) Cells were transfected with an empty vector (pcDNA3, as a control) or dominant negative mutant of p38 MAPK (Δp38) or JNK (ΔJNK) for 24 h, and then exposed to TGF-β1 for 16 h. (F) For cell migration, cells were pretreated with U0126 (10 μM) or SP600125 (10 μM) for 1 h and then incubated with TGF-β1 (15 ng/ml) for 48 h. The image is representative of three similar experiments (n = 3). The conditioned media (A, B, E) were analyzed gelatin zymorgraphy, and the whole cell lysates (D) were analyzed using an anti-phospho-JNK1/2 or anti-GAPDH (as an internal control) antibody. Data are expressed as mean ± SEM (D) or mean (A, B, C, E) of at least three independent experiments (n = 3). * P

Techniques Used: Expressing, Reverse Transcription Polymerase Chain Reaction, Incubation, Transfection, Plasmid Preparation, Dominant Negative Mutation, Migration

10) Product Images from "Artocarpin, an isoprenyl flavonoid, induces p53-dependent or independent apoptosis via ROS-mediated MAPKs and Akt activation in non-small cell lung cancer cells"

Article Title: Artocarpin, an isoprenyl flavonoid, induces p53-dependent or independent apoptosis via ROS-mediated MAPKs and Akt activation in non-small cell lung cancer cells

Journal: Oncotarget

doi: 10.18632/oncotarget.16058

ROS-dependent MAPKs activation is involved in artocarpin-induced apoptosis in A549 and H1299 cells Cells were treated with 10 μM artocarpin for different periods of time, followed by Western blot analysis using antibodies against phospho-p38 MAPK, phospho-ERK1/2 or phospho-Akt. ( A ) Western blot analysis showing the effects of 10 μM SB202190 (p38 inhibitor), 10 μM U0126 (ERK1/2 inhibitor), 10 μM LY294002 (PI3K inhibitor), APO (100 μM) or NAC (100 μM) on the phosphorylation of p38 MAPK, ERK1/2 or Akt in artocarpin-treated A549 and H1299 cells. The blots were probed for GAPDH as a loading control. ( B ) Cross talk between MAPKs and Akt phosphorylation in artocarpin-treated A549 and H1299 cells. Cells were pretreated with 10 μM SB202190, 10 μM U0126 or 10 μM LY294002 for 1hr and then treated with 10 μM artocarpin for 4 hr. ( C ) Relationship between MAPKs, Akt and p47 phox in artocarpin-treated A549 and H1299 cells. Cells were transfected with either scrambled siRNA or p47 phox siRNA and then treated with 10 μM artocarpin for 4 hr. ( D , E ) Cells were pretreated with10 μM SB202190, 10 μM U0126 or 10 μM LY294002 for 1 h, and then treated with 10 μM artocarpin for 24h. Cytotoxicity was determined using MTT assay and stained with Annexin-V and PI as previously described in Figure 1 . Data shown are means ± SEM. * P
Figure Legend Snippet: ROS-dependent MAPKs activation is involved in artocarpin-induced apoptosis in A549 and H1299 cells Cells were treated with 10 μM artocarpin for different periods of time, followed by Western blot analysis using antibodies against phospho-p38 MAPK, phospho-ERK1/2 or phospho-Akt. ( A ) Western blot analysis showing the effects of 10 μM SB202190 (p38 inhibitor), 10 μM U0126 (ERK1/2 inhibitor), 10 μM LY294002 (PI3K inhibitor), APO (100 μM) or NAC (100 μM) on the phosphorylation of p38 MAPK, ERK1/2 or Akt in artocarpin-treated A549 and H1299 cells. The blots were probed for GAPDH as a loading control. ( B ) Cross talk between MAPKs and Akt phosphorylation in artocarpin-treated A549 and H1299 cells. Cells were pretreated with 10 μM SB202190, 10 μM U0126 or 10 μM LY294002 for 1hr and then treated with 10 μM artocarpin for 4 hr. ( C ) Relationship between MAPKs, Akt and p47 phox in artocarpin-treated A549 and H1299 cells. Cells were transfected with either scrambled siRNA or p47 phox siRNA and then treated with 10 μM artocarpin for 4 hr. ( D , E ) Cells were pretreated with10 μM SB202190, 10 μM U0126 or 10 μM LY294002 for 1 h, and then treated with 10 μM artocarpin for 24h. Cytotoxicity was determined using MTT assay and stained with Annexin-V and PI as previously described in Figure 1 . Data shown are means ± SEM. * P

Techniques Used: Activation Assay, Western Blot, Transfection, MTT Assay, Staining

Artocarpin up-regulated the expression of p53-dependent apoptotic proteins via p38 MAPK and ERK1/2 pathway, but not by Akt pathway A549 cells were treated with 10 μM artocarpin for various periods of time, followed by Western blot analysis using antibodies against phospho-p53, PUMA, Cytochrome C, Apaf-1and caspase-3. Western blot analysis also demonstrating the effects of 10 μM SB202190, 10 μM U0126, 10 μM LY294002, APO (100 μM) or NAC (100 μM) treatment on the up-regulation of phospho-p53, PUMA, Cytochrome C, Apaf-1and caspase-3 in artocarpin-treated A549 cells. The blots were probed for GAPDH as a loading control. Each blot is representative of three independent experiments, all of which had similar results.
Figure Legend Snippet: Artocarpin up-regulated the expression of p53-dependent apoptotic proteins via p38 MAPK and ERK1/2 pathway, but not by Akt pathway A549 cells were treated with 10 μM artocarpin for various periods of time, followed by Western blot analysis using antibodies against phospho-p53, PUMA, Cytochrome C, Apaf-1and caspase-3. Western blot analysis also demonstrating the effects of 10 μM SB202190, 10 μM U0126, 10 μM LY294002, APO (100 μM) or NAC (100 μM) treatment on the up-regulation of phospho-p53, PUMA, Cytochrome C, Apaf-1and caspase-3 in artocarpin-treated A549 cells. The blots were probed for GAPDH as a loading control. Each blot is representative of three independent experiments, all of which had similar results.

Techniques Used: Expressing, Western Blot

11) Product Images from "Mitogen-Activated Protein Kinases Inhibit the ROMK (Kir 1.1)-Like Small Conductance K Channels in the Cortical Collecting Duct"

Article Title: Mitogen-Activated Protein Kinases Inhibit the ROMK (Kir 1.1)-Like Small Conductance K Channels in the Cortical Collecting Duct

Journal: Journal of the American Society of Nephrology : JASN

doi: 10.1681/ASN.2006050426

Effect of PD098059+SB202190 on the SK channels in the presence and absence of herbimycin A in the CCD from rats that were on a normal K diet. The experiments were performed in cell-attached patches. Herbimycin A (1 µM) was used to treated the CCD for 15 min and present throughout the experiment. * P
Figure Legend Snippet: Effect of PD098059+SB202190 on the SK channels in the presence and absence of herbimycin A in the CCD from rats that were on a normal K diet. The experiments were performed in cell-attached patches. Herbimycin A (1 µM) was used to treated the CCD for 15 min and present throughout the experiment. * P

Techniques Used:

(A) Effect of H 2 O 2 on the expression of c-Src in M-1 cells in the absence of MAPK inhibitors and in the presence of PD098059 (50 µM), SP600125 (10 µM), SB202190 (5 µM), PD+SB, or three inhibitors. Data are summarized and presented as bar graph at the bottom of the figure. ** P
Figure Legend Snippet: (A) Effect of H 2 O 2 on the expression of c-Src in M-1 cells in the absence of MAPK inhibitors and in the presence of PD098059 (50 µM), SP600125 (10 µM), SB202190 (5 µM), PD+SB, or three inhibitors. Data are summarized and presented as bar graph at the bottom of the figure. ** P

Techniques Used: Expressing

A channel recording showing that simultaneous inhibition of ERK with PD098059 (PD) and P38 MAPK with SB202190 (SB) activates the small-conductance K (SK) channels in the cortical collecting duct (CCD) from a rat that was on a normal K diet. The experiments were performed in cell-attached patches, and the holding potential was 30 mV. The top trace is the time course of the experiment, whereas three parts of trace, indicated by numbers, are extended to show the fast time resolution. The channel close level is indicated by “C.” The arrow indicates the addition of MAPK inhibitors that were present throughout the experiments. Also, the electric noise after addition of PD+SB was artifact.
Figure Legend Snippet: A channel recording showing that simultaneous inhibition of ERK with PD098059 (PD) and P38 MAPK with SB202190 (SB) activates the small-conductance K (SK) channels in the cortical collecting duct (CCD) from a rat that was on a normal K diet. The experiments were performed in cell-attached patches, and the holding potential was 30 mV. The top trace is the time course of the experiment, whereas three parts of trace, indicated by numbers, are extended to show the fast time resolution. The channel close level is indicated by “C.” The arrow indicates the addition of MAPK inhibitors that were present throughout the experiments. Also, the electric noise after addition of PD+SB was artifact.

Techniques Used: Inhibition

12) Product Images from "TNF-α Mediates PKCδ/JNK1/2/c-Jun-Dependent Monocyte Adhesion via ICAM-1 Induction in Human Retinal Pigment Epithelial Cells"

Article Title: TNF-α Mediates PKCδ/JNK1/2/c-Jun-Dependent Monocyte Adhesion via ICAM-1 Induction in Human Retinal Pigment Epithelial Cells

Journal: PLoS ONE

doi: 10.1371/journal.pone.0117911

TNF-α induces ICAM-1 expression via a PKCδ/JNK1/2 signaling in human RPECs. (A) Cells were pretreated with U0126, SP600125, or SB202190 for 1 h, and then incubated with TNF-α for 6 h. The protein expression of ICAM-1 was determined by Western blot. (B) Cells were transfected with siRNA of scrambled, JNK2, or p42, and then incubated with TNF-α for 6 h. The protein levels of p42, JNK2, and ICAM-1 were determined by Western blot. (C) Cells were pretreated with U0126 (10 μM), SP600125 (10 μM), or SB202190 (10 μM) for 1 h, and then incubated with TNF-α for 4 h or 6 h. The mRNA levels and promoter activity of ICAM-1 were determined by real-time PCR and promoter assay, respectively. (D) Cells were pretreated without or with U0126 or SP600125 for 1 h, and then incubated with TNF-α for the indicated times. The levels of phospho-p42/p44 MAPK and phospho-JNK1/2 were determined by Western blot. (E) Cells were pretreated without or with Rottlerin for 1 h, and then incubated with TNF-α for the indicated time intervals. The levels of phospho-p42/p44 MAPK and phospho-JNK1/2 were determined by Western blot. (F) RPECs were pretreated with Ro318220, Rottlerin, SP600125, or U0126 for 1 h, and then incubated with TNF-α for 6 h. The THP-1 cells adherence was measured. Data are expressed as mean±S.E.M. of three independent experiments. * P
Figure Legend Snippet: TNF-α induces ICAM-1 expression via a PKCδ/JNK1/2 signaling in human RPECs. (A) Cells were pretreated with U0126, SP600125, or SB202190 for 1 h, and then incubated with TNF-α for 6 h. The protein expression of ICAM-1 was determined by Western blot. (B) Cells were transfected with siRNA of scrambled, JNK2, or p42, and then incubated with TNF-α for 6 h. The protein levels of p42, JNK2, and ICAM-1 were determined by Western blot. (C) Cells were pretreated with U0126 (10 μM), SP600125 (10 μM), or SB202190 (10 μM) for 1 h, and then incubated with TNF-α for 4 h or 6 h. The mRNA levels and promoter activity of ICAM-1 were determined by real-time PCR and promoter assay, respectively. (D) Cells were pretreated without or with U0126 or SP600125 for 1 h, and then incubated with TNF-α for the indicated times. The levels of phospho-p42/p44 MAPK and phospho-JNK1/2 were determined by Western blot. (E) Cells were pretreated without or with Rottlerin for 1 h, and then incubated with TNF-α for the indicated time intervals. The levels of phospho-p42/p44 MAPK and phospho-JNK1/2 were determined by Western blot. (F) RPECs were pretreated with Ro318220, Rottlerin, SP600125, or U0126 for 1 h, and then incubated with TNF-α for 6 h. The THP-1 cells adherence was measured. Data are expressed as mean±S.E.M. of three independent experiments. * P

Techniques Used: Expressing, Incubation, Western Blot, Transfection, Activity Assay, Real-time Polymerase Chain Reaction, Promoter Assay

13) Product Images from "Urban particulate matter down-regulates filaggrin via COX2 expression/PGE2 production leading to skin barrier dysfunction"

Article Title: Urban particulate matter down-regulates filaggrin via COX2 expression/PGE2 production leading to skin barrier dysfunction

Journal: Scientific Reports

doi: 10.1038/srep27995

PMs induce COX2 expression via Nox2/ERK/p38/NF-κB signaling. Cells were pre-incubated with NF-κB inhibitor (Bay11-7082; BAY) (10 μM) for 1 h and then exposed to PMs (50 μg/cm 2 ) for 24 h. ( A) COX2 protein expression (open bars) and prostaglandin E2 (PGE2) production (shaded bars) were determined by Western blotting and ELISA as described in Fig. 1 . ( B,C ) Cells were pre-incubated with with AhRI (10 μM), APO (100 μM), U0126 (10 μM), SB202190 (10 μM), SP60015 (10 μM) or BAY (10 μM) for 1 h and then exposed to PMs (50 μg/cm 2 ) for the 2 h. The levels of p65 in nuclear extract (detected with p65 or phospho-p65 antibodies), determined by Western blotting (C) . Lamin-B1 was used as the loading control. ( D) Cells were pre-incubated with with U0126 (10 μM), SB202190 (10 μM), SP60015 (10 μM) or BAY (10 μM) for 1 h and then exposed to PMs (50 μg/cm 2 ) for 2 h. Electrophoretic mobility-shift assay for assessment of NF-κB DNA binding activity, as described in “Materials and methods”. ( A–D ) Data are expressed as mean ± standard error of the mean, based on three independent experiments. * P
Figure Legend Snippet: PMs induce COX2 expression via Nox2/ERK/p38/NF-κB signaling. Cells were pre-incubated with NF-κB inhibitor (Bay11-7082; BAY) (10 μM) for 1 h and then exposed to PMs (50 μg/cm 2 ) for 24 h. ( A) COX2 protein expression (open bars) and prostaglandin E2 (PGE2) production (shaded bars) were determined by Western blotting and ELISA as described in Fig. 1 . ( B,C ) Cells were pre-incubated with with AhRI (10 μM), APO (100 μM), U0126 (10 μM), SB202190 (10 μM), SP60015 (10 μM) or BAY (10 μM) for 1 h and then exposed to PMs (50 μg/cm 2 ) for the 2 h. The levels of p65 in nuclear extract (detected with p65 or phospho-p65 antibodies), determined by Western blotting (C) . Lamin-B1 was used as the loading control. ( D) Cells were pre-incubated with with U0126 (10 μM), SB202190 (10 μM), SP60015 (10 μM) or BAY (10 μM) for 1 h and then exposed to PMs (50 μg/cm 2 ) for 2 h. Electrophoretic mobility-shift assay for assessment of NF-κB DNA binding activity, as described in “Materials and methods”. ( A–D ) Data are expressed as mean ± standard error of the mean, based on three independent experiments. * P

Techniques Used: Expressing, Incubation, Western Blot, Enzyme-linked Immunosorbent Assay, Electrophoretic Mobility Shift Assay, Binding Assay, Activity Assay

Reactive oxygen species (ROS)-dependent MAPK activation is involved in PMs-induced up-regulation of COX2 expression in HaCaT cells. Cells were pre-incubated with AhRI (10 μM), APO (100 μM), U0126 (10 μM), SB202190 (10 μM), SP60015 (10 μM) or DPI (10 μM) for 1 h and then exposed to PMs (50 μg/cm 2 ) for the indicated times (0, 30′, 1 h, 2 h, 4 h and 6 h). ( A–C ) Effects of MAPK inhibitors and antioxidants on phosphorylation of p-ERK1/2 ( A ) p-JNK1/2 ( B ) and p-p38 ( C ). Total levels of each kinase were used as loading controls. Effects of siRNAs or inhibitors of ERK1/2, JNK1/2 and p38 on the phosphorylation of MAPKs ( D ) COX2 protein expression ( E ) COX2 gene expression ( F ) and prostaglandin E2 (PGE2) production ( G ). To assess gene expression ( F ), cells were co-transfected with a COX2 reporter gene and following addition of siRNAs, exposed to PMs for 4 h (for promoter activity evaluations; open bars) or 6 h (for evaluations of mRNA levels; shaded bars). Representative results from three separate experiments are shown. * P
Figure Legend Snippet: Reactive oxygen species (ROS)-dependent MAPK activation is involved in PMs-induced up-regulation of COX2 expression in HaCaT cells. Cells were pre-incubated with AhRI (10 μM), APO (100 μM), U0126 (10 μM), SB202190 (10 μM), SP60015 (10 μM) or DPI (10 μM) for 1 h and then exposed to PMs (50 μg/cm 2 ) for the indicated times (0, 30′, 1 h, 2 h, 4 h and 6 h). ( A–C ) Effects of MAPK inhibitors and antioxidants on phosphorylation of p-ERK1/2 ( A ) p-JNK1/2 ( B ) and p-p38 ( C ). Total levels of each kinase were used as loading controls. Effects of siRNAs or inhibitors of ERK1/2, JNK1/2 and p38 on the phosphorylation of MAPKs ( D ) COX2 protein expression ( E ) COX2 gene expression ( F ) and prostaglandin E2 (PGE2) production ( G ). To assess gene expression ( F ), cells were co-transfected with a COX2 reporter gene and following addition of siRNAs, exposed to PMs for 4 h (for promoter activity evaluations; open bars) or 6 h (for evaluations of mRNA levels; shaded bars). Representative results from three separate experiments are shown. * P

Techniques Used: Activation Assay, Expressing, Incubation, Transfection, Activity Assay

Activation of activator protein 1 (AP-1) is a critical event in PMs-induced up-regulation of COX2 expression. Cells were pre-incubated with AP-1 inhibitor (tanshinone IIA; Tan) (10 μM) for 1 h and then exposed to PMs (50 μg/cm 2 ) for 24 h. ( A) COX2 protein expression (open bars) and prostaglandin E2 (PGE2) production (shaded bars) were determined by Western blotting and ELISA as described in Fig. 1 . (B,C ) Cells were pre-incubated with AhRI (10 μM), APO (100 μM), U0126 (10 μM), SB202190 (10 μM), SP60015 (10 μM) or BAY (10 μM) for 1 h and then exposed to PMs (50 μg/cm 2 ) for 2 h. The nuclear extract levels of c-Jun and c-Fos protein were detected using phospho-c-Jun and phospho-c-Fos antibodies, determined by Western blotting (Fig. 6 C ). Lamin-B1 was used as the loading control. ( D) Cells were pre-incubated with U0126 (10 μM), SB202190 (10 μM), SP60015 (10 μM) or Tan (10 μM) for 1 h and then exposed to PMs (50 μg/cm 2 ) for 2 h. Electrophoretic mobility-shift assay for assessment of AP-1 DNA binding activity, as described in “Materials and methods”. Data are expressed as mean ± standard error of the mean, based on three independent experiments. * P
Figure Legend Snippet: Activation of activator protein 1 (AP-1) is a critical event in PMs-induced up-regulation of COX2 expression. Cells were pre-incubated with AP-1 inhibitor (tanshinone IIA; Tan) (10 μM) for 1 h and then exposed to PMs (50 μg/cm 2 ) for 24 h. ( A) COX2 protein expression (open bars) and prostaglandin E2 (PGE2) production (shaded bars) were determined by Western blotting and ELISA as described in Fig. 1 . (B,C ) Cells were pre-incubated with AhRI (10 μM), APO (100 μM), U0126 (10 μM), SB202190 (10 μM), SP60015 (10 μM) or BAY (10 μM) for 1 h and then exposed to PMs (50 μg/cm 2 ) for 2 h. The nuclear extract levels of c-Jun and c-Fos protein were detected using phospho-c-Jun and phospho-c-Fos antibodies, determined by Western blotting (Fig. 6 C ). Lamin-B1 was used as the loading control. ( D) Cells were pre-incubated with U0126 (10 μM), SB202190 (10 μM), SP60015 (10 μM) or Tan (10 μM) for 1 h and then exposed to PMs (50 μg/cm 2 ) for 2 h. Electrophoretic mobility-shift assay for assessment of AP-1 DNA binding activity, as described in “Materials and methods”. Data are expressed as mean ± standard error of the mean, based on three independent experiments. * P

Techniques Used: Activation Assay, Expressing, Incubation, Western Blot, Enzyme-linked Immunosorbent Assay, Electrophoretic Mobility Shift Assay, Binding Assay, Activity Assay

14) Product Images from "Hypertonic Stress Induces VEGF Production in Human Colon Cancer Cell Line Caco-2: Inhibitory Role of Autocrine PGE2"

Article Title: Hypertonic Stress Induces VEGF Production in Human Colon Cancer Cell Line Caco-2: Inhibitory Role of Autocrine PGE2

Journal: PLoS ONE

doi: 10.1371/journal.pone.0025193

Role of MAPKs in VEGF production by Caco-2 cells. Inhibitors of JNK, SP600125; p38, SB202190; and MEK 1/2, U0126 were added before stimulation with hypertonic stress (100 mM NaCl) ( A ) or 1 mM CoCl 2 ( B ) for 24 h. Caco-2 cells were pretreated with 1 µM of NS-398 to prevent endogenous PGE 2 production in all samples. VEGF production was determined by ELISA in supernatants of Caco-2 cells. +, * p
Figure Legend Snippet: Role of MAPKs in VEGF production by Caco-2 cells. Inhibitors of JNK, SP600125; p38, SB202190; and MEK 1/2, U0126 were added before stimulation with hypertonic stress (100 mM NaCl) ( A ) or 1 mM CoCl 2 ( B ) for 24 h. Caco-2 cells were pretreated with 1 µM of NS-398 to prevent endogenous PGE 2 production in all samples. VEGF production was determined by ELISA in supernatants of Caco-2 cells. +, * p

Techniques Used: Enzyme-linked Immunosorbent Assay

15) Product Images from "Sphingosine 1-Phosphate Induces Cyclooxygenase-2/Prostaglandin E2 Expression via PKCα-dependent Mitogen-Activated Protein Kinases and NF-κB Cascade in Human Cardiac Fibroblasts"

Article Title: Sphingosine 1-Phosphate Induces Cyclooxygenase-2/Prostaglandin E2 Expression via PKCα-dependent Mitogen-Activated Protein Kinases and NF-κB Cascade in Human Cardiac Fibroblasts

Journal: Frontiers in Pharmacology

doi: 10.3389/fphar.2020.569802

NF-κB is essential for S1P-induced COX-2 expression. (A) Cells were pretreated with helenalin for 1 h and then incubated with 15 μM S1P for 8 h. (B) Cells were transfected without or with COX-2 promoter-luciferase reporter gene, pretreated without or with helenalin (1 μM) for 1 h, and then incubated with 15 μM S1P for 4 h (mRNA) or 1 h (promoter). The levels of COX-2 protein, mRNA expression, and promoter activity were determined by Western blot, real time-PCR, and promoter assay, respectively. (C) Cells were transfected with siRNA of p65 and then exposed to S1P for 8 h. (D) Cells were incubated with S1P (15 μM) for the indicated time intervals. (E) Cells were treated with S1P (15 μM) for 15 min in the absence or presence of helenalin (1 μM). (D,E) The nuclear and cytosol fractions were prepared and analyzed by Western blot. The p65 NF-κB translocation by S1P was also determined by immunofluorescent staining as described in Materials and Methods . (F) To determine which S1PR subtypes, Gi or Gq protein, and MAPKs involved in S1P-stimulated the nuclear localization of p65 NF-κB, cells were incubated with S1P (15 μM) for 15 min in the absence or presence of Gö6976 (Gö, 10 μM), SB202190 (SB, 30 μM), SP600125 (SP, 10 μM), PD98059 (PD, 10 μM), W123 (W, 10 μM), CAY (10 μM), GPA2A (GPA, 10 μM), or PTX (100 ng/ml), and the nuclear fraction was analyzed by Western blot. To fit the construct of data layout, the data were rearranged from the same gel with the exception of non-related inhibitors and disclosed by the insertion of white spaces rearranged from the original capture. Data are expressed as mean ± SEM of three individual experiments (n = 3). # p
Figure Legend Snippet: NF-κB is essential for S1P-induced COX-2 expression. (A) Cells were pretreated with helenalin for 1 h and then incubated with 15 μM S1P for 8 h. (B) Cells were transfected without or with COX-2 promoter-luciferase reporter gene, pretreated without or with helenalin (1 μM) for 1 h, and then incubated with 15 μM S1P for 4 h (mRNA) or 1 h (promoter). The levels of COX-2 protein, mRNA expression, and promoter activity were determined by Western blot, real time-PCR, and promoter assay, respectively. (C) Cells were transfected with siRNA of p65 and then exposed to S1P for 8 h. (D) Cells were incubated with S1P (15 μM) for the indicated time intervals. (E) Cells were treated with S1P (15 μM) for 15 min in the absence or presence of helenalin (1 μM). (D,E) The nuclear and cytosol fractions were prepared and analyzed by Western blot. The p65 NF-κB translocation by S1P was also determined by immunofluorescent staining as described in Materials and Methods . (F) To determine which S1PR subtypes, Gi or Gq protein, and MAPKs involved in S1P-stimulated the nuclear localization of p65 NF-κB, cells were incubated with S1P (15 μM) for 15 min in the absence or presence of Gö6976 (Gö, 10 μM), SB202190 (SB, 30 μM), SP600125 (SP, 10 μM), PD98059 (PD, 10 μM), W123 (W, 10 μM), CAY (10 μM), GPA2A (GPA, 10 μM), or PTX (100 ng/ml), and the nuclear fraction was analyzed by Western blot. To fit the construct of data layout, the data were rearranged from the same gel with the exception of non-related inhibitors and disclosed by the insertion of white spaces rearranged from the original capture. Data are expressed as mean ± SEM of three individual experiments (n = 3). # p

Techniques Used: Expressing, Incubation, Transfection, Luciferase, Activity Assay, Western Blot, Real-time Polymerase Chain Reaction, Promoter Assay, Translocation Assay, Staining, Construct

COX-2 promoter activity is stimulated by S1P through an NF-κB-dependent pathway. (A) Cells were incubated with S1P for the indicated time intervals (upper panel). Cells were pretreated with PD98059 (10 μM), SB202190 (30 μM), SP600125 (10 μM) for 1 h and then incubated with S1P for 30 min. The binding activity of NF-κB p65 and promoter was analyzed by ChIP assay (n = 3), as described in Methods . Left panel, to fit the construct of data layout, the data were rearranged from the same gel with the exception of non-related inhibitors and disclosed by the insertion of white spaces rearranged from the original capture. (B,C) Cells were transfected with an NF-κB-luciferase reporter gene, pretreated with W123 (10 μM), CAY (10 μM), GPA2A (10 μM), PTX (100 ng/ml), Gö6976 (10 μM), PD98059 (10 μM), SB202190 (30 μM), SP600125 (10 μM), or helenalin (1 μM) for 1 h and then incubated with S1P for 2 h. (D) The schematic picture represented two different 5′-promoter regions of the mouse COX-2 promoter constructs, both wild-type (WT) and mt-κB modified by single-point mutation of the κB binding site fused to the pGL-luciferase reporter gene. “↲ ” indicated the translational start site (+1) of the luciferase reporter gene. WT COX-2 promoter reporter gene (WT-COX-2) or NF-κB mutated COX-2 promoter reporter gene (mt-κB-COX-2) were transfected into cells, which then were incubated with or without S1P for 1 h. The promoter reporter activity was determined. (E) Cells were pretreated with W123, CAY, GPA2A, PTX, Gö6976, PD98059, SB202190, SP600125, or helenalin for 1 h and then incubated with S1P for 8 h. The PGE 2 levels were analyzed by EIA. Data are expressed as mean ± SEM of three individual experiments (n = 3). # p
Figure Legend Snippet: COX-2 promoter activity is stimulated by S1P through an NF-κB-dependent pathway. (A) Cells were incubated with S1P for the indicated time intervals (upper panel). Cells were pretreated with PD98059 (10 μM), SB202190 (30 μM), SP600125 (10 μM) for 1 h and then incubated with S1P for 30 min. The binding activity of NF-κB p65 and promoter was analyzed by ChIP assay (n = 3), as described in Methods . Left panel, to fit the construct of data layout, the data were rearranged from the same gel with the exception of non-related inhibitors and disclosed by the insertion of white spaces rearranged from the original capture. (B,C) Cells were transfected with an NF-κB-luciferase reporter gene, pretreated with W123 (10 μM), CAY (10 μM), GPA2A (10 μM), PTX (100 ng/ml), Gö6976 (10 μM), PD98059 (10 μM), SB202190 (30 μM), SP600125 (10 μM), or helenalin (1 μM) for 1 h and then incubated with S1P for 2 h. (D) The schematic picture represented two different 5′-promoter regions of the mouse COX-2 promoter constructs, both wild-type (WT) and mt-κB modified by single-point mutation of the κB binding site fused to the pGL-luciferase reporter gene. “↲ ” indicated the translational start site (+1) of the luciferase reporter gene. WT COX-2 promoter reporter gene (WT-COX-2) or NF-κB mutated COX-2 promoter reporter gene (mt-κB-COX-2) were transfected into cells, which then were incubated with or without S1P for 1 h. The promoter reporter activity was determined. (E) Cells were pretreated with W123, CAY, GPA2A, PTX, Gö6976, PD98059, SB202190, SP600125, or helenalin for 1 h and then incubated with S1P for 8 h. The PGE 2 levels were analyzed by EIA. Data are expressed as mean ± SEM of three individual experiments (n = 3). # p

Techniques Used: Activity Assay, Incubation, Binding Assay, Chromatin Immunoprecipitation, Construct, Transfection, Luciferase, Modification, Mutagenesis, Enzyme-linked Immunosorbent Assay

S1P-induced COX-2 expression is mediated through p38 MAPK phosphorylation. (A) Cells pretreated with SB202190 for 1 h were followed by incubation with 15 μM S1P for 8 h. (B) Cells were transfected without or with COX-2 promoter-luciferase reporter gene, pretreated without or with SB202190 (30 μM) for 1 h, and then incubated with 15 μM S1P for 4 h (mRNA) or 1 h (promoter). The levels of COX-2 protein, mRNA expression, and promoter activity were individually examined by Western blot, real time-PCR, and promoter assay. (C) Cells transfected with p38α MAPK siRNA were incubated with S1P for 8 h. ( D ) Cells were incubated with S1P (15 μM) for the indicated time intervals in the absence or presence of SB202190 (30 μM), W123 (10 μM), CAY (10 μM), GPA2A (10 μM), or Gö6976 (10 μM) for 1 h and PTX (100 ng/ml) for 24 h. The cell lysates were collected and analyzed by Western blot. The fold of basal was defined as normalization of the data to the respective “0,” and then compared the data of corresponding time points of control vs inhibitor with a statistic method, as described in the section of Methods . Data are expressed as mean ± SEM of three individual experiments (n = 3). # p
Figure Legend Snippet: S1P-induced COX-2 expression is mediated through p38 MAPK phosphorylation. (A) Cells pretreated with SB202190 for 1 h were followed by incubation with 15 μM S1P for 8 h. (B) Cells were transfected without or with COX-2 promoter-luciferase reporter gene, pretreated without or with SB202190 (30 μM) for 1 h, and then incubated with 15 μM S1P for 4 h (mRNA) or 1 h (promoter). The levels of COX-2 protein, mRNA expression, and promoter activity were individually examined by Western blot, real time-PCR, and promoter assay. (C) Cells transfected with p38α MAPK siRNA were incubated with S1P for 8 h. ( D ) Cells were incubated with S1P (15 μM) for the indicated time intervals in the absence or presence of SB202190 (30 μM), W123 (10 μM), CAY (10 μM), GPA2A (10 μM), or Gö6976 (10 μM) for 1 h and PTX (100 ng/ml) for 24 h. The cell lysates were collected and analyzed by Western blot. The fold of basal was defined as normalization of the data to the respective “0,” and then compared the data of corresponding time points of control vs inhibitor with a statistic method, as described in the section of Methods . Data are expressed as mean ± SEM of three individual experiments (n = 3). # p

Techniques Used: Expressing, Incubation, Transfection, Luciferase, Activity Assay, Western Blot, Real-time Polymerase Chain Reaction, Promoter Assay

16) Product Images from "Subanesthetic isoflurane relieves zymosan-induced neutrophil inflammatory response by targeting NMDA glutamate receptor and Toll-like receptor 2 signaling"

Article Title: Subanesthetic isoflurane relieves zymosan-induced neutrophil inflammatory response by targeting NMDA glutamate receptor and Toll-like receptor 2 signaling

Journal: Oncotarget

doi: 10.18632/oncotarget.9091

Isoflurane and zymosan regulate NF-κB activation via ROS/p38 MAPK signaling A. , B. Neutrophils were sequentially treated with zymosan (10 μg/ml) for 15 min, 0.7% isoflurane for 15 min where indicated, and zymosan for 6 h in the presence or absence of NAC (50 mM). Cells were subjected to fluorescence measurement of H 2 O 2 levels (A) and Western blot analysis (B). Scale bar = 10 μM. C. , D. Neutrophils were treated with zymosan for 6 h in the presence of the p38 MAPK inhibitor SB202190 (10 mM) where indicated, followed by immunofluorescent staining of p65 (C) and Western blot analysis of nuclear p65 levels (D). Scale bar = 10 μM. E. , F. Neutrophils were treated with zymosan for indicated time periods, followed by measurement of NADPH oxidase activity (E) or Western blot assay using the membrane and cytosol fractions of cell lysates (F). Gas and β-actin were used as inner controls for membrane and cytosolic fractions, respectively. G. Neutrophils were treated with zymosan for 12 h in the presense of NAC (50 mM), SB202190 (10 mM) or NADPH oxidase inhibitor DPI (10 mM) where indicated, followed by measurement of NO and ONOO − production. Data are represented as the mean ± SEM of 3 replicates or representative of 3 independent experiments. * P
Figure Legend Snippet: Isoflurane and zymosan regulate NF-κB activation via ROS/p38 MAPK signaling A. , B. Neutrophils were sequentially treated with zymosan (10 μg/ml) for 15 min, 0.7% isoflurane for 15 min where indicated, and zymosan for 6 h in the presence or absence of NAC (50 mM). Cells were subjected to fluorescence measurement of H 2 O 2 levels (A) and Western blot analysis (B). Scale bar = 10 μM. C. , D. Neutrophils were treated with zymosan for 6 h in the presence of the p38 MAPK inhibitor SB202190 (10 mM) where indicated, followed by immunofluorescent staining of p65 (C) and Western blot analysis of nuclear p65 levels (D). Scale bar = 10 μM. E. , F. Neutrophils were treated with zymosan for indicated time periods, followed by measurement of NADPH oxidase activity (E) or Western blot assay using the membrane and cytosol fractions of cell lysates (F). Gas and β-actin were used as inner controls for membrane and cytosolic fractions, respectively. G. Neutrophils were treated with zymosan for 12 h in the presense of NAC (50 mM), SB202190 (10 mM) or NADPH oxidase inhibitor DPI (10 mM) where indicated, followed by measurement of NO and ONOO − production. Data are represented as the mean ± SEM of 3 replicates or representative of 3 independent experiments. * P

Techniques Used: Activation Assay, Fluorescence, Western Blot, Staining, Activity Assay

17) Product Images from "Inhibition of MAPK stimulates the Ca2+-dependent big-conductance K channels in cortical collecting duct"

Article Title: Inhibition of MAPK stimulates the Ca2+-dependent big-conductance K channels in cortical collecting duct

Journal:

doi: 10.1073/pnas.0609555104

A channel recording showing the effect of inhibiting ERK with PD 098059 and P38 MAPK with SB202190 on BK channels in a cell-attached patch of PC. The top trace shows the time course of the experiment, and the three parts of the data indicated by numbers
Figure Legend Snippet: A channel recording showing the effect of inhibiting ERK with PD 098059 and P38 MAPK with SB202190 on BK channels in a cell-attached patch of PC. The top trace shows the time course of the experiment, and the three parts of the data indicated by numbers

Techniques Used:

A bar graph shows the probability of finding BK channels identified in PC and IC ( A ) and channel activity (NP o ) ( B ) in the absence (left set of bars) or presence (middle set of bars) of PD098059+SB202190 or in the CCD from rats on a high K diet (right
Figure Legend Snippet: A bar graph shows the probability of finding BK channels identified in PC and IC ( A ) and channel activity (NP o ) ( B ) in the absence (left set of bars) or presence (middle set of bars) of PD098059+SB202190 or in the CCD from rats on a high K diet (right

Techniques Used: Activity Assay

A channel recording showing the effect of inhibiting P38 MAPK with SB202190 on BK channels in a cell-attached patch of PC. The top trace shows the time course of the experiment, and the three parts of the data indicated by numbers are demonstrated in
Figure Legend Snippet: A channel recording showing the effect of inhibiting P38 MAPK with SB202190 on BK channels in a cell-attached patch of PC. The top trace shows the time course of the experiment, and the three parts of the data indicated by numbers are demonstrated in

Techniques Used:

18) Product Images from "Sphingosine-1-Phosphate Mediates ICAM-1-Dependent Monocyte Adhesion through p38 MAPK and p42/p44 MAPK-Dependent Akt Activation"

Article Title: Sphingosine-1-Phosphate Mediates ICAM-1-Dependent Monocyte Adhesion through p38 MAPK and p42/p44 MAPK-Dependent Akt Activation

Journal: PLoS ONE

doi: 10.1371/journal.pone.0118473

S1P induces ICAM-1 expression via MAPKs. (A) HPAEpiCs were pretreated with U0126, SB202190, or SP600125 for 1 h, and then incubated with S1P for 16 h. The ICAM-1 protein expression was determined by Western blot. (B) Cells were pretreated with SB202190 (10 M), U0126 (1 μM), or SP600125 (10 M) for 1 h, and then incubated with S1P for 4 h. The ICAM-1 mRNA expression and promoter activity were determined by real-time PCR and promoter assay, respectively. (C) Cells were pretreated with SB202190 (10 M), U0126 (1 μM), or SP600125 (10 M) for 1 h, and then incubated with S1P for 16 h. The THP-1 cells adherence was measured. (D) Cells were transfected with siRNA of scrambled, p38, p42, or JNK1, and then incubated with S1P (10 μM) for 16 h. The levels of p38, p42, JNK1, and ICAM-1 proteins were determined by Western blot. (E) Cells were pretreated without or with U0126 (1 μM), SB202190 (10 M), or SP600125 (10 M) for 1 h, and then incubated with S1P (10 M) for the indicated time intervals. The levels of phospho-p42/p44 MAPK, phospho-p38 MAPK, and phospho-JNK1/2 were determined by Western blot. Data are expressed as mean (A, D) or mean±S.E.M (B, C) of three independent experiments. * P
Figure Legend Snippet: S1P induces ICAM-1 expression via MAPKs. (A) HPAEpiCs were pretreated with U0126, SB202190, or SP600125 for 1 h, and then incubated with S1P for 16 h. The ICAM-1 protein expression was determined by Western blot. (B) Cells were pretreated with SB202190 (10 M), U0126 (1 μM), or SP600125 (10 M) for 1 h, and then incubated with S1P for 4 h. The ICAM-1 mRNA expression and promoter activity were determined by real-time PCR and promoter assay, respectively. (C) Cells were pretreated with SB202190 (10 M), U0126 (1 μM), or SP600125 (10 M) for 1 h, and then incubated with S1P for 16 h. The THP-1 cells adherence was measured. (D) Cells were transfected with siRNA of scrambled, p38, p42, or JNK1, and then incubated with S1P (10 μM) for 16 h. The levels of p38, p42, JNK1, and ICAM-1 proteins were determined by Western blot. (E) Cells were pretreated without or with U0126 (1 μM), SB202190 (10 M), or SP600125 (10 M) for 1 h, and then incubated with S1P (10 M) for the indicated time intervals. The levels of phospho-p42/p44 MAPK, phospho-p38 MAPK, and phospho-JNK1/2 were determined by Western blot. Data are expressed as mean (A, D) or mean±S.E.M (B, C) of three independent experiments. * P

Techniques Used: Expressing, Incubation, Western Blot, Activity Assay, Real-time Polymerase Chain Reaction, Promoter Assay, Transfection

S1P induces ICAM-1 expression via AP-1. (A) HPAEpiCs were pretreated with Tanshinone IIA for 1 h, and then incubated with S1P for 16 h. The ICAM-1 protein expression was determined by Western blot. (B) Cells were pretreated with Tanshinone IIA (100 nM) for 1 h, and then incubated with S1P for 4 h. The ICAM-1 mRNA expression and promoter activity were determined by real-time PCR and promoter assay, respectively. (C) Cells were pretreated with Tanshinone IIA (100 nM) for 1 h, and then incubated with S1P for 16 h. The THP-1 cells adherence was measured. (D) Cells were treated with S1P for the indicated time intervals. The mRNA levels of c-Jun and c-Fos were determined by real-time PCR. (E) Cells were transfected with siRNA of scrambled, c-Jun, or c-Fos, and then incubated with S1P (10 μM) for 16 h. The levels of c-Fos, c-Jun, and ICAM-1 proteins were determined by Western blot. (F) Cells were pretreated without or with PP1, AG1296, AG1478, U0126, SP600125, SB202190, or U0126 for 1 h, and then incubated with S1P for the indicated time intervals. The levels of phospho-c-Jun were determined by Western blot. Data are expressed as mean (A, E) or mean±S.E.M. of three independent experiments. # P
Figure Legend Snippet: S1P induces ICAM-1 expression via AP-1. (A) HPAEpiCs were pretreated with Tanshinone IIA for 1 h, and then incubated with S1P for 16 h. The ICAM-1 protein expression was determined by Western blot. (B) Cells were pretreated with Tanshinone IIA (100 nM) for 1 h, and then incubated with S1P for 4 h. The ICAM-1 mRNA expression and promoter activity were determined by real-time PCR and promoter assay, respectively. (C) Cells were pretreated with Tanshinone IIA (100 nM) for 1 h, and then incubated with S1P for 16 h. The THP-1 cells adherence was measured. (D) Cells were treated with S1P for the indicated time intervals. The mRNA levels of c-Jun and c-Fos were determined by real-time PCR. (E) Cells were transfected with siRNA of scrambled, c-Jun, or c-Fos, and then incubated with S1P (10 μM) for 16 h. The levels of c-Fos, c-Jun, and ICAM-1 proteins were determined by Western blot. (F) Cells were pretreated without or with PP1, AG1296, AG1478, U0126, SP600125, SB202190, or U0126 for 1 h, and then incubated with S1P for the indicated time intervals. The levels of phospho-c-Jun were determined by Western blot. Data are expressed as mean (A, E) or mean±S.E.M. of three independent experiments. # P

Techniques Used: Expressing, Incubation, Western Blot, Activity Assay, Real-time Polymerase Chain Reaction, Promoter Assay, Transfection

S1P induces ICAM-1 expression via PI3K/Akt. (A) HPAEpiCs were pretreated with LY294002 for 1 h, and then incubated with S1P for 16 h. The ICAM-1 protein expression was determined by Western blot. (B) Cells were pretreated with LY294002 (10 M) for 1 h, and then incubated with S1P for 4 h. The ICAM-1 mRNA expression and promoter activity were determined by real-time PCR and promoter assay, respectively. (C) Cells were pretreated with AG1478 (10 M), AG1296 (10 μM), or LY294002 (10 μM) for 1 h, and then incubated with S1P for 16 h. The THP-1 cells adherence was measured. (D-F) Cells were pretreated with LY294002, Genistein, PP1, AG1478, AG1296, SB202190, SP600125, or U0126 for 1 h, and then incubated with S1P for the indicated time intervals. The levels of phospho-Akt were determined by Western blot. Data are expressed as mean±S.E.M. of three independent experiments. # P
Figure Legend Snippet: S1P induces ICAM-1 expression via PI3K/Akt. (A) HPAEpiCs were pretreated with LY294002 for 1 h, and then incubated with S1P for 16 h. The ICAM-1 protein expression was determined by Western blot. (B) Cells were pretreated with LY294002 (10 M) for 1 h, and then incubated with S1P for 4 h. The ICAM-1 mRNA expression and promoter activity were determined by real-time PCR and promoter assay, respectively. (C) Cells were pretreated with AG1478 (10 M), AG1296 (10 μM), or LY294002 (10 μM) for 1 h, and then incubated with S1P for 16 h. The THP-1 cells adherence was measured. (D-F) Cells were pretreated with LY294002, Genistein, PP1, AG1478, AG1296, SB202190, SP600125, or U0126 for 1 h, and then incubated with S1P for the indicated time intervals. The levels of phospho-Akt were determined by Western blot. Data are expressed as mean±S.E.M. of three independent experiments. # P

Techniques Used: Expressing, Incubation, Western Blot, Activity Assay, Real-time Polymerase Chain Reaction, Promoter Assay

19) Product Images from "Up-regulation of COX-2/PGE2 by endothelin-1 via MAPK-dependent NF-?B pathway in mouse brain microvascular endothelial cells"

Article Title: Up-regulation of COX-2/PGE2 by endothelin-1 via MAPK-dependent NF-?B pathway in mouse brain microvascular endothelial cells

Journal: Cell Communication and Signaling : CCS

doi: 10.1186/1478-811X-11-8

NF-κB (p65) is essential for ET1-1-induced COX-2 expression. ( A, B ) Cells were treated with 10 nM ET-1 for 6 h in the absence or presence of Bay11-7082. The COX-2 protein and mRNA expression were determined by Western blot and RT-PCR as described in Figure 1 . ( C ) Time dependence of ET-1-stimulated p65 NF-κB translocation by subcellular isolation, Western blot, and immunofluorescent stain. ( D ) Cells were pretreated with U0126 (1 μM), SB202190 (300 nM), SP600125 (300 nM), BQ-788 (1 μM), or Bay11-7082 (10 nM) for 1 h and then incubated with ET-1 (10 nM) for 90 min. The nuclear fraction was analyzed by Western blot. ( E ) Cells were transfected with p65 siRNA and then exposed to ET-1 for 6 h. The cell lysates were collected and analyzed by Western blotting using an anti-COX-2, anti-p65, anti-Lamin A, or anti-GAPDH (as an internal control) antibody. Data are expressed as mean ± SEM of at least three individual experiments (n=3 in each group; # P
Figure Legend Snippet: NF-κB (p65) is essential for ET1-1-induced COX-2 expression. ( A, B ) Cells were treated with 10 nM ET-1 for 6 h in the absence or presence of Bay11-7082. The COX-2 protein and mRNA expression were determined by Western blot and RT-PCR as described in Figure 1 . ( C ) Time dependence of ET-1-stimulated p65 NF-κB translocation by subcellular isolation, Western blot, and immunofluorescent stain. ( D ) Cells were pretreated with U0126 (1 μM), SB202190 (300 nM), SP600125 (300 nM), BQ-788 (1 μM), or Bay11-7082 (10 nM) for 1 h and then incubated with ET-1 (10 nM) for 90 min. The nuclear fraction was analyzed by Western blot. ( E ) Cells were transfected with p65 siRNA and then exposed to ET-1 for 6 h. The cell lysates were collected and analyzed by Western blotting using an anti-COX-2, anti-p65, anti-Lamin A, or anti-GAPDH (as an internal control) antibody. Data are expressed as mean ± SEM of at least three individual experiments (n=3 in each group; # P

Techniques Used: Expressing, Western Blot, Reverse Transcription Polymerase Chain Reaction, Translocation Assay, Isolation, Staining, Incubation, Transfection

ET-1-induced COX-2 expression is mediated through MAPKs phosphorylation. ( A, B ) Cells were treated with 10 nM ET-1 for ( A ) 6 h and ( B ) 1 h in the absence or presence of U0126, SB202190, or SP600125. The COX-2 protein and mRNA expression were determined by Western blot and RT-PCR. ( C ) Time dependence of ET-1-stimulated ERK1/2, p38 MAPK, and JNK1/2 phosphorylation, cells were incubated with 10 nM ET-1 for the indicated times in the absence or presence of U0126 (1 μM), SB202190 (300 nM), or SP600125 (300 nM). ( D ) Cells were transfected with siRNA of ERK2, p38 MAPK, or JNK1 and then exposed to ET-1 for 6 h. ( E ) Cells were pretreated with BQ-788 (1 μM), GPA2 (1 μM), or GPA2A (1 μM) for 1 h and then incubated with ET-1 (10 nM) for the indicated times. The cell lysates were collected and analyzed by Western blotting using an anti-COX-2, anti-phospho-ERK1/2, anti-phospho-p38 MAPK, anti-phospho-JNK1/2, anti-ERK2, anti-p38 MAPK, anti-JNK1, or anti-GAPDH (as an internal control) antibody. Data are expressed as mean ± SEM of at least three individual experiments (n=3 in each group; * P
Figure Legend Snippet: ET-1-induced COX-2 expression is mediated through MAPKs phosphorylation. ( A, B ) Cells were treated with 10 nM ET-1 for ( A ) 6 h and ( B ) 1 h in the absence or presence of U0126, SB202190, or SP600125. The COX-2 protein and mRNA expression were determined by Western blot and RT-PCR. ( C ) Time dependence of ET-1-stimulated ERK1/2, p38 MAPK, and JNK1/2 phosphorylation, cells were incubated with 10 nM ET-1 for the indicated times in the absence or presence of U0126 (1 μM), SB202190 (300 nM), or SP600125 (300 nM). ( D ) Cells were transfected with siRNA of ERK2, p38 MAPK, or JNK1 and then exposed to ET-1 for 6 h. ( E ) Cells were pretreated with BQ-788 (1 μM), GPA2 (1 μM), or GPA2A (1 μM) for 1 h and then incubated with ET-1 (10 nM) for the indicated times. The cell lysates were collected and analyzed by Western blotting using an anti-COX-2, anti-phospho-ERK1/2, anti-phospho-p38 MAPK, anti-phospho-JNK1/2, anti-ERK2, anti-p38 MAPK, anti-JNK1, or anti-GAPDH (as an internal control) antibody. Data are expressed as mean ± SEM of at least three individual experiments (n=3 in each group; * P

Techniques Used: Expressing, Western Blot, Reverse Transcription Polymerase Chain Reaction, Incubation, Transfection

ET-1-stimulated COX-2 promoter activity is mediated through NF-κB-dependent pathway. ( A ) Time dependence of ET-1-enhanced NF-κB transcription activity, cells were transfected with a NF-κB-luciferase reporter gene and then exposed to ET-1 for the indicated times. ( B ) After transfection, the cells were pretreated with BQ-788 (1 μM), GPA2 (1 μM), GPA2A (1 μM), U0126 (1 μM), SB202190 (300 nM), SP600125 (300 nM), or ( A ) Bay11-7082 (10 nM) for 1 h and then incubated with ET-1 (10 nM) for 60 min. ( C ) Cells were pretreated with BQ-788, U0126, SB202190, or SP600125 for 1 h and then incubated with ET-1. The p65 NF-κB binding activity was analyzed by ChIP-PCR as described in Methods. ( D ) For COX-2 promoter activity, cells were transfected with a COX-2-promoter-luciferase reporter gene and then exposed to ET-1. After transfection, the cells were pretreated with BQ-788, GPA2, GPA2A, U0126, SB202190, SP600125, or Bay11-7082 for 1 h and then incubated with ET-1 for 6 h. ( E ) Schematic representation of a 5′-promoter regions of the mouse different COX-2 promoter constructs, either wild-type (WT) or mutation of the κB-binding site (mt-κB) fused to the pGL-luciferase reporter gene, the translational start site (+1) of the luciferase reporter gene was indicated by an arrow. Cells were transfected with WT COX-2 promoter reporter gene (WT-COX-2) or NF-κB mutated COX-2 promoter reporter gene (mt-κB-COX-2) and then incubated with or without ET-1 for 6 h. The promoter reporter assay was performed as described in Methods. ( F ) Cells were pretreated with BQ-123, BQ-788, GPA2, GPA2A, U0126, SB202190, SP600125, Bay11-7082, or transfected with p65 siRNA and then incubated with ET-1 for 6 h. The PGE 2 levels were analyzed by EIA. Data are expressed as mean ± SEM of at least three individual experiments (n=3 in each group; # P
Figure Legend Snippet: ET-1-stimulated COX-2 promoter activity is mediated through NF-κB-dependent pathway. ( A ) Time dependence of ET-1-enhanced NF-κB transcription activity, cells were transfected with a NF-κB-luciferase reporter gene and then exposed to ET-1 for the indicated times. ( B ) After transfection, the cells were pretreated with BQ-788 (1 μM), GPA2 (1 μM), GPA2A (1 μM), U0126 (1 μM), SB202190 (300 nM), SP600125 (300 nM), or ( A ) Bay11-7082 (10 nM) for 1 h and then incubated with ET-1 (10 nM) for 60 min. ( C ) Cells were pretreated with BQ-788, U0126, SB202190, or SP600125 for 1 h and then incubated with ET-1. The p65 NF-κB binding activity was analyzed by ChIP-PCR as described in Methods. ( D ) For COX-2 promoter activity, cells were transfected with a COX-2-promoter-luciferase reporter gene and then exposed to ET-1. After transfection, the cells were pretreated with BQ-788, GPA2, GPA2A, U0126, SB202190, SP600125, or Bay11-7082 for 1 h and then incubated with ET-1 for 6 h. ( E ) Schematic representation of a 5′-promoter regions of the mouse different COX-2 promoter constructs, either wild-type (WT) or mutation of the κB-binding site (mt-κB) fused to the pGL-luciferase reporter gene, the translational start site (+1) of the luciferase reporter gene was indicated by an arrow. Cells were transfected with WT COX-2 promoter reporter gene (WT-COX-2) or NF-κB mutated COX-2 promoter reporter gene (mt-κB-COX-2) and then incubated with or without ET-1 for 6 h. The promoter reporter assay was performed as described in Methods. ( F ) Cells were pretreated with BQ-123, BQ-788, GPA2, GPA2A, U0126, SB202190, SP600125, Bay11-7082, or transfected with p65 siRNA and then incubated with ET-1 for 6 h. The PGE 2 levels were analyzed by EIA. Data are expressed as mean ± SEM of at least three individual experiments (n=3 in each group; # P

Techniques Used: Activity Assay, Transfection, Luciferase, Incubation, Binding Assay, Chromatin Immunoprecipitation, Polymerase Chain Reaction, Construct, Mutagenesis, Reporter Assay, Enzyme-linked Immunosorbent Assay

20) Product Images from "Role of TLR4/NADPH oxidase/ROS-activated p38 MAPK in VCAM-1 expression induced by lipopolysaccharide in human renal mesangial cells"

Article Title: Role of TLR4/NADPH oxidase/ROS-activated p38 MAPK in VCAM-1 expression induced by lipopolysaccharide in human renal mesangial cells

Journal: Cell Communication and Signaling : CCS

doi: 10.1186/1478-811X-10-33

LPS induces VCAM-1 expression via ROS/p38 MAPK. ( A ) Cells were pretreated with SB202190, U0126, or SP600125, and then incubated with LPS for 8 h. VCAM-1 protein levels were determined. ( B ) Cells were pretreated with SB202190, U0126, or SP600125, and then incubated with LPS for 4 h or 6 h. mRNA levels and promoter activity of VCAM-1 were determined. ( C ) Cells were treated with LPS for the indicated times or transfected with siRNA of c-Src, p47 phox , or p38 MAPK, and then treated with LPS for 1 h. Phospho-p38 MAPK expression was determined. ( D ) Cells were treated with LPS for the indicated times or pretreated with edaravone (Eda, 100 μM), PP1 (1 μM), or U0126 (U0, 1 μM), and then treated with LPS for 5 min. Phospho-p42/p44 MAPK expression was determined. ( E ) Cells were treated with LPS for the indicated times or pretreated with edaravone (Eda), PP1, or SP600125 (SP), and then treated with LPS for 1 h. The expression of phospho-JNK1/2 was determined. ( F ) Cells were transfected with p38 MAPK siRNA, and then incubated with LPS for 8 h. Protein levels of p38 MAPK and VCAM-1 were determined. All figures are representative of three independent experiments, performed in duplicate. Data are expressed as means ± SEM. * P
Figure Legend Snippet: LPS induces VCAM-1 expression via ROS/p38 MAPK. ( A ) Cells were pretreated with SB202190, U0126, or SP600125, and then incubated with LPS for 8 h. VCAM-1 protein levels were determined. ( B ) Cells were pretreated with SB202190, U0126, or SP600125, and then incubated with LPS for 4 h or 6 h. mRNA levels and promoter activity of VCAM-1 were determined. ( C ) Cells were treated with LPS for the indicated times or transfected with siRNA of c-Src, p47 phox , or p38 MAPK, and then treated with LPS for 1 h. Phospho-p38 MAPK expression was determined. ( D ) Cells were treated with LPS for the indicated times or pretreated with edaravone (Eda, 100 μM), PP1 (1 μM), or U0126 (U0, 1 μM), and then treated with LPS for 5 min. Phospho-p42/p44 MAPK expression was determined. ( E ) Cells were treated with LPS for the indicated times or pretreated with edaravone (Eda), PP1, or SP600125 (SP), and then treated with LPS for 1 h. The expression of phospho-JNK1/2 was determined. ( F ) Cells were transfected with p38 MAPK siRNA, and then incubated with LPS for 8 h. Protein levels of p38 MAPK and VCAM-1 were determined. All figures are representative of three independent experiments, performed in duplicate. Data are expressed as means ± SEM. * P

Techniques Used: Expressing, Incubation, Activity Assay, Transfection

LPS induces VCAM-1 expression via p300. Cells were pretreated with GR343, and then incubated with LPS for ( A ) 8 h or ( B ) 4 h. Levels of VCAM-1 protein ( A ) and mRNA ( B ) expression were determined. ( C ) Cells were pretreated with 1 μM GR343, and then incubated with LPS for 6 h. VCAM-1 promoter activity was determined. ( D ) Cells were transfected with p300 siRNA, and then incubated with LPS for 8 h. Protein levels of p300 and VCAM-1 were determined. ( E ) Cells were treated with LPS for the indicated times. The cell lysates were subjected to Western blot using an anti-phospho-p300 antibody. ( F ) Cells were pretreated with GR343 (GR, 1 μM), PP1 (1 μM), MCI-186 (MCI, 100 μM), APO (100 μM), or SB202190 (SB, 1 μM), and then treated with LPS for 90 min. The cell lysates were subjected to Western blot using an anti-phospho-p300 antibody. ( G ) Cells were treated with LPS for the indicated times. The cell lysates were subjected to immunoprecipitation using an anti-p300 antibody, and then the immunoprecipitates were analyzed by Western blot using an anti-ATF2 or anti-p300 antibody. All figures are representative of three independent experiments, performed in duplicate. Data are expressed as means ± SEM. * P
Figure Legend Snippet: LPS induces VCAM-1 expression via p300. Cells were pretreated with GR343, and then incubated with LPS for ( A ) 8 h or ( B ) 4 h. Levels of VCAM-1 protein ( A ) and mRNA ( B ) expression were determined. ( C ) Cells were pretreated with 1 μM GR343, and then incubated with LPS for 6 h. VCAM-1 promoter activity was determined. ( D ) Cells were transfected with p300 siRNA, and then incubated with LPS for 8 h. Protein levels of p300 and VCAM-1 were determined. ( E ) Cells were treated with LPS for the indicated times. The cell lysates were subjected to Western blot using an anti-phospho-p300 antibody. ( F ) Cells were pretreated with GR343 (GR, 1 μM), PP1 (1 μM), MCI-186 (MCI, 100 μM), APO (100 μM), or SB202190 (SB, 1 μM), and then treated with LPS for 90 min. The cell lysates were subjected to Western blot using an anti-phospho-p300 antibody. ( G ) Cells were treated with LPS for the indicated times. The cell lysates were subjected to immunoprecipitation using an anti-p300 antibody, and then the immunoprecipitates were analyzed by Western blot using an anti-ATF2 or anti-p300 antibody. All figures are representative of three independent experiments, performed in duplicate. Data are expressed as means ± SEM. * P

Techniques Used: Expressing, Incubation, Activity Assay, Transfection, Western Blot, Immunoprecipitation

21) Product Images from "c-Src-dependent EGF receptor transactivation contributes to ET-1-induced COX-2 expression in brain microvascular endothelial cells"

Article Title: c-Src-dependent EGF receptor transactivation contributes to ET-1-induced COX-2 expression in brain microvascular endothelial cells

Journal: Journal of Neuroinflammation

doi: 10.1186/1742-2094-9-152

ET-1 stimulates c-Src-dependent transactivation of EGFR/PI3K/Akt leading to MAPKs and c-Jun phosphorylation. ( A ) For c-Src phosphorylation, cells were incubated with 10 nM ET-1 for the indicated time intervals in the absence or presence of BQ788 (1 μM), GPAnt2 (1 μM), GPAnt2A (1 μM), AG1478 (1 μM), or LY294002 (300 nM). ( B ) For EGFR phosphorylation, cells were incubated with 10 nM ET-1 for the indicated time intervals in the absence or presence of BQ788, GPAnt2, GPAnt2A, or PP1 (100 nM). ( C ) For Akt phosphorylation, cells were incubated with 10 nM ET-1 for the indicated time intervals in the absence or presence of BQ788, GPAnt2, GPAnt2A, PP1, AG1478, or LY294002. ( D ) For MAPKs phosphorylation, cells were incubated with 10 nM ET-1 for the indicated time intervals in the absence or presence of PP1, AG1478, or LY294002. For COX-2 expression, cells were incubated with ET-1 (10 nM) for 6 h in the absence or presence of U0126 (1 μM), SB202190 (300 nM), or SP600125 (300 nM). ( E ) For c-Jun phosphorylation, cells were incubated with 10 nM ET-1 for the indicated time intervals in the absence or presence of BQ788, GPAnt2, GPAnt2A, PP1, AG1478, LY294002, U0126 (1 μM), SB202190 (300 nM), or SP600125 (300 nM). The cell lysates were collected and analyzed by Western blotting using an anti-phospho-c-Src, anti-phospho-EGFR, anti-phospho-Akt, anti-phospho-ERK1/2, anti-phospho-p38 MAPK, anti-phospho-JNK1/2, anti-phospho-c-Jun, or anti-GAPDH (as an internal control) antibody. The figure represents one of three similar experiments ( n = 3).
Figure Legend Snippet: ET-1 stimulates c-Src-dependent transactivation of EGFR/PI3K/Akt leading to MAPKs and c-Jun phosphorylation. ( A ) For c-Src phosphorylation, cells were incubated with 10 nM ET-1 for the indicated time intervals in the absence or presence of BQ788 (1 μM), GPAnt2 (1 μM), GPAnt2A (1 μM), AG1478 (1 μM), or LY294002 (300 nM). ( B ) For EGFR phosphorylation, cells were incubated with 10 nM ET-1 for the indicated time intervals in the absence or presence of BQ788, GPAnt2, GPAnt2A, or PP1 (100 nM). ( C ) For Akt phosphorylation, cells were incubated with 10 nM ET-1 for the indicated time intervals in the absence or presence of BQ788, GPAnt2, GPAnt2A, PP1, AG1478, or LY294002. ( D ) For MAPKs phosphorylation, cells were incubated with 10 nM ET-1 for the indicated time intervals in the absence or presence of PP1, AG1478, or LY294002. For COX-2 expression, cells were incubated with ET-1 (10 nM) for 6 h in the absence or presence of U0126 (1 μM), SB202190 (300 nM), or SP600125 (300 nM). ( E ) For c-Jun phosphorylation, cells were incubated with 10 nM ET-1 for the indicated time intervals in the absence or presence of BQ788, GPAnt2, GPAnt2A, PP1, AG1478, LY294002, U0126 (1 μM), SB202190 (300 nM), or SP600125 (300 nM). The cell lysates were collected and analyzed by Western blotting using an anti-phospho-c-Src, anti-phospho-EGFR, anti-phospho-Akt, anti-phospho-ERK1/2, anti-phospho-p38 MAPK, anti-phospho-JNK1/2, anti-phospho-c-Jun, or anti-GAPDH (as an internal control) antibody. The figure represents one of three similar experiments ( n = 3).

Techniques Used: Incubation, Expressing, Western Blot

ET-1-induced PGE 2 release is mediated through a c-Src-dependent transactivation of EGFR/PI3K/Akt cascade. ( A ) Cells were incubated with 10 nM ET-1 for 6 h in the absence or presence of genistein (Gen, 10 nM), PP1 (100 nM), AG1478 (AG, 1 μM), LY294002 (LY, 00 nM), SH-5 (100 nM), U0126 (U0, 1 μM), SB202190 (SB, 300 nM), or SP600125 (SP, 300 nM), and the conditioned media were collected to assay the PGE 2 level by EIA kit. Data are expressed as mean ± SEM of three individual experiments ( n = 3). # P
Figure Legend Snippet: ET-1-induced PGE 2 release is mediated through a c-Src-dependent transactivation of EGFR/PI3K/Akt cascade. ( A ) Cells were incubated with 10 nM ET-1 for 6 h in the absence or presence of genistein (Gen, 10 nM), PP1 (100 nM), AG1478 (AG, 1 μM), LY294002 (LY, 00 nM), SH-5 (100 nM), U0126 (U0, 1 μM), SB202190 (SB, 300 nM), or SP600125 (SP, 300 nM), and the conditioned media were collected to assay the PGE 2 level by EIA kit. Data are expressed as mean ± SEM of three individual experiments ( n = 3). # P

Techniques Used: Incubation, Enzyme-linked Immunosorbent Assay

ET-1-stimulated COX-2 promoter activity is mediated through AP-1-dependent pathway. ( A ) Time dependence of ET-1-enhanced AP-1 transcription activity; cells were transfected with an AP-1-luciferase reporter gene and then exposed to ET-1 for the indicated time intervals. ( B ) After transfection with AP-1-luciferase reporter gene, the cells were pretreated with PP1 PP1 (100 nM), AG1478 (AG, 1 μM), LY294002 (LY, 300 nM), SH-5 (10 nM), U0126 (U0, 1 μM), SB202190 (SB, 300 nM), SP600125 (SP, 300 nM), or ( A ) TSIIA (100 nM) for 1 h and then incubated with ET-1 (10 nM) for 90 min. ( C ) Cells were pretreated without or with TSIIA, U0126 (U0), SB202190 (SB), SP600125 (SP), or BQ788 (BQ) for 1 h and then incubated with ET-1 (10 nM) for the indicated time intervals (upper panel) or 90 min (lower panel). The c-Jun/AP-1 binding activity was analyzed by chromatin-IP (ChIP)-PCR assay. ( D ) For COX-2 promoter activity, cells were pretreated with PP1, AG1478 (AG), LY294002 (LY), SH-5, U0126 (U0), SB202190 (SB), or SP600125 (SP), or TSIIA for 1 h and then incubated with ET-1 (10 nM) for 6 h. ( E ) Schematic representation of a 5′-promoter regions of the mouse different COX-2 promoter constructs, either wild-type (WT) or mutated by single-point mutation of the AP-1 binding site (mu-AP-1) cloned to the pGL-luciferase reporter gene; the translational start site (+1) of the luciferase reporter gene is indicated by an arrow. Cells were transfected with WT COX-2 promoter reporter gene (WT) or AP-1 mutated COX-2 promoter reporter gene (mu-AP-1) and then incubated with or without ET-1 (10 nM) for 6 h. The promoter reporter activity was determined. Data are expressed as mean ± SEM of at least three individual experiments ( n = 3). * P
Figure Legend Snippet: ET-1-stimulated COX-2 promoter activity is mediated through AP-1-dependent pathway. ( A ) Time dependence of ET-1-enhanced AP-1 transcription activity; cells were transfected with an AP-1-luciferase reporter gene and then exposed to ET-1 for the indicated time intervals. ( B ) After transfection with AP-1-luciferase reporter gene, the cells were pretreated with PP1 PP1 (100 nM), AG1478 (AG, 1 μM), LY294002 (LY, 300 nM), SH-5 (10 nM), U0126 (U0, 1 μM), SB202190 (SB, 300 nM), SP600125 (SP, 300 nM), or ( A ) TSIIA (100 nM) for 1 h and then incubated with ET-1 (10 nM) for 90 min. ( C ) Cells were pretreated without or with TSIIA, U0126 (U0), SB202190 (SB), SP600125 (SP), or BQ788 (BQ) for 1 h and then incubated with ET-1 (10 nM) for the indicated time intervals (upper panel) or 90 min (lower panel). The c-Jun/AP-1 binding activity was analyzed by chromatin-IP (ChIP)-PCR assay. ( D ) For COX-2 promoter activity, cells were pretreated with PP1, AG1478 (AG), LY294002 (LY), SH-5, U0126 (U0), SB202190 (SB), or SP600125 (SP), or TSIIA for 1 h and then incubated with ET-1 (10 nM) for 6 h. ( E ) Schematic representation of a 5′-promoter regions of the mouse different COX-2 promoter constructs, either wild-type (WT) or mutated by single-point mutation of the AP-1 binding site (mu-AP-1) cloned to the pGL-luciferase reporter gene; the translational start site (+1) of the luciferase reporter gene is indicated by an arrow. Cells were transfected with WT COX-2 promoter reporter gene (WT) or AP-1 mutated COX-2 promoter reporter gene (mu-AP-1) and then incubated with or without ET-1 (10 nM) for 6 h. The promoter reporter activity was determined. Data are expressed as mean ± SEM of at least three individual experiments ( n = 3). * P

Techniques Used: Activity Assay, Transfection, Luciferase, Incubation, Binding Assay, Chromatin Immunoprecipitation, Polymerase Chain Reaction, Construct, Mutagenesis, Clone Assay

22) Product Images from "NADPH Oxidase/ROS-Dependent VCAM-1 Induction on TNF-α-Challenged Human Cardiac Fibroblasts Enhances Monocyte Adhesion"

Article Title: NADPH Oxidase/ROS-Dependent VCAM-1 Induction on TNF-α-Challenged Human Cardiac Fibroblasts Enhances Monocyte Adhesion

Journal: Frontiers in Pharmacology

doi: 10.3389/fphar.2015.00310

Up-regulated VCAM-1 by TNF-α contributes to promotion of monocyte adhering to these HCFs. (A) For cell adhesion activity, cells were pretreated with VCAM-1 nAb, TNFR nAb, Gö6976 (Gö), DPI, edaravone (Eda) ( Figure 7A , upper), U0126 (U0), SB202190 (SB), SP600125 (SP), or TSIIA ( Figure 7A , lower) for 1 h and following by TNF-α (15 μg/ml) for 16 h prior to addition of THP-1 monocytes. The adhesion activity was determined by cell adhesion assay as described in Section “Materials and Methods”. Data are expressed as mean ± SEM of at least five individual experiments ( n = 5). # P
Figure Legend Snippet: Up-regulated VCAM-1 by TNF-α contributes to promotion of monocyte adhering to these HCFs. (A) For cell adhesion activity, cells were pretreated with VCAM-1 nAb, TNFR nAb, Gö6976 (Gö), DPI, edaravone (Eda) ( Figure 7A , upper), U0126 (U0), SB202190 (SB), SP600125 (SP), or TSIIA ( Figure 7A , lower) for 1 h and following by TNF-α (15 μg/ml) for 16 h prior to addition of THP-1 monocytes. The adhesion activity was determined by cell adhesion assay as described in Section “Materials and Methods”. Data are expressed as mean ± SEM of at least five individual experiments ( n = 5). # P

Techniques Used: Activity Assay, Cell Adhesion Assay

Involvement of p38 MAPK in TNF-α-induced VCAM-1 expression in HCFs. (A) HCFs were pretreated with SB202190 (0.1, 1, or 10 μM) for 1 h, and then incubated with TNF-α for 16 h. The protein levels of VCAM-1 were determined by Western blot. (B) Cells were pretreated with SB202190 (10 μM) for 1 h, and then incubated with TNF-α for 4 h. The mRNA expression (white bar) and promoter activity (gray bar) of VCAM-1 were determined by real-time PCR or promoter report assay, respectively. (C) Cells were pretreated with SB202190 (10 μM) for 1 h, and then incubated with TNF-α for the indicated time intervals. The levels of phospho-p38 MAPK were determined by Western blot. (D) Cells were transfected with either scrambled or p38 MAPK siRNA, and then incubated with TNF-α for 16 h. The levels of p38 MAPK and VCAM-1 protein were determined by Western blot. (E) Cells were pretreated without or with TNFR nAb (10 μg/ml), Gö6976 (10 μM), edaravone (10 μM), or DPI (10 μM) for 1 h, and then incubated with TNF-α for the indicated time intervals. The levels of phospho-p38 MAPK were determined by Western blot. Data are expressed as mean ± SEM of three independent experiments ( n = 3, Quantitative data of Figures 5C–E were presented in Supplementary Table S1 ). # P
Figure Legend Snippet: Involvement of p38 MAPK in TNF-α-induced VCAM-1 expression in HCFs. (A) HCFs were pretreated with SB202190 (0.1, 1, or 10 μM) for 1 h, and then incubated with TNF-α for 16 h. The protein levels of VCAM-1 were determined by Western blot. (B) Cells were pretreated with SB202190 (10 μM) for 1 h, and then incubated with TNF-α for 4 h. The mRNA expression (white bar) and promoter activity (gray bar) of VCAM-1 were determined by real-time PCR or promoter report assay, respectively. (C) Cells were pretreated with SB202190 (10 μM) for 1 h, and then incubated with TNF-α for the indicated time intervals. The levels of phospho-p38 MAPK were determined by Western blot. (D) Cells were transfected with either scrambled or p38 MAPK siRNA, and then incubated with TNF-α for 16 h. The levels of p38 MAPK and VCAM-1 protein were determined by Western blot. (E) Cells were pretreated without or with TNFR nAb (10 μg/ml), Gö6976 (10 μM), edaravone (10 μM), or DPI (10 μM) for 1 h, and then incubated with TNF-α for the indicated time intervals. The levels of phospho-p38 MAPK were determined by Western blot. Data are expressed as mean ± SEM of three independent experiments ( n = 3, Quantitative data of Figures 5C–E were presented in Supplementary Table S1 ). # P

Techniques Used: Expressing, Incubation, Western Blot, Activity Assay, Real-time Polymerase Chain Reaction, Transfection

Activation of c-Jun/AP-1 is required for TNF-α-mediated VCAM-1 expression. (A) HCFs were pretreated with TSIIA (0.01, 0.1, or 1 μM) for 1 h, and then incubated with TNF-α for 16 h. The protein levels of VCAM-1 were determined by Western blot. (B) HCFs were pretreated with TSIIA (1 μM) for 1 h, and then incubated with TNF-α for 4 h. The mRNA expression (white bar) and promoter activity (gray bar) of VCAM-1 were determined by real-time PCR or promoter report assay, respectively. (C) HCFs were pretreated with TSIIA (1 μM) for 1 h, and then incubated with TNF-α for the indicated time intervals. The levels of phospho-c-Jun were determined by Western blot. (D) HCFs were transfected with either scrambled or c-Jun siRNA, and then incubated with TNF-α for 16 h. The levels of c-Jun and VCAM-1 protein were determined by Western blot. (E) HCFs were pretreated without or with TNFR nAb (10 μg/ml), Gö6976 (10 μM), edaravone (10 μM), DPI (10 μM), U0126 (1 μM), SP600125 (3 μM) for 1 h, or p38 siRNA transfection, and then incubated with TNF-α for the indicated time intervals. The levels of phospho-c-Jun were determined by Western blot. (F) HCFs were transiently cotransfected with pAP1-Luc and pCMV-Gal for 24 h, and then incubated with TNF-α for the indicated time intervals ( F , left). The transfected cells were pretreated with TNFR nAb, Gö6976, DPI, edaravone (Eda) ( F , middle), apocynin (APO), SB202190 (SB), SP600125 (SP), or TSIIA ( F , right) for 1 h and then incubated with TNF-α for 2 h. The AP-1 transcription activity in the cell was determined as described in Section “Materials and Methods”. Data are expressed as mean ± SEM of three independent experiments ( n = 3, Quantitative data of Figures 7C–E were presented in Supplementary Table S2 ). ∗ P
Figure Legend Snippet: Activation of c-Jun/AP-1 is required for TNF-α-mediated VCAM-1 expression. (A) HCFs were pretreated with TSIIA (0.01, 0.1, or 1 μM) for 1 h, and then incubated with TNF-α for 16 h. The protein levels of VCAM-1 were determined by Western blot. (B) HCFs were pretreated with TSIIA (1 μM) for 1 h, and then incubated with TNF-α for 4 h. The mRNA expression (white bar) and promoter activity (gray bar) of VCAM-1 were determined by real-time PCR or promoter report assay, respectively. (C) HCFs were pretreated with TSIIA (1 μM) for 1 h, and then incubated with TNF-α for the indicated time intervals. The levels of phospho-c-Jun were determined by Western blot. (D) HCFs were transfected with either scrambled or c-Jun siRNA, and then incubated with TNF-α for 16 h. The levels of c-Jun and VCAM-1 protein were determined by Western blot. (E) HCFs were pretreated without or with TNFR nAb (10 μg/ml), Gö6976 (10 μM), edaravone (10 μM), DPI (10 μM), U0126 (1 μM), SP600125 (3 μM) for 1 h, or p38 siRNA transfection, and then incubated with TNF-α for the indicated time intervals. The levels of phospho-c-Jun were determined by Western blot. (F) HCFs were transiently cotransfected with pAP1-Luc and pCMV-Gal for 24 h, and then incubated with TNF-α for the indicated time intervals ( F , left). The transfected cells were pretreated with TNFR nAb, Gö6976, DPI, edaravone (Eda) ( F , middle), apocynin (APO), SB202190 (SB), SP600125 (SP), or TSIIA ( F , right) for 1 h and then incubated with TNF-α for 2 h. The AP-1 transcription activity in the cell was determined as described in Section “Materials and Methods”. Data are expressed as mean ± SEM of three independent experiments ( n = 3, Quantitative data of Figures 7C–E were presented in Supplementary Table S2 ). ∗ P

Techniques Used: Activation Assay, Expressing, Incubation, Western Blot, Activity Assay, Real-time Polymerase Chain Reaction, Transfection

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    Biomol GmbH sb202190
    Isoflurane and zymosan regulate NF-κB activation via ROS/p38 MAPK signaling A. , B. Neutrophils were sequentially treated with zymosan (10 μg/ml) for 15 min, 0.7% isoflurane for 15 min where indicated, and zymosan for 6 h in the presence or absence of NAC (50 mM). Cells were subjected to fluorescence measurement of H 2 O 2 levels (A) and Western blot analysis (B). Scale bar = 10 μM. C. , D. Neutrophils were treated with zymosan for 6 h in the presence of the p38 MAPK inhibitor <t>SB202190</t> (10 mM) where indicated, followed by immunofluorescent staining of p65 (C) and Western blot analysis of nuclear p65 levels (D). Scale bar = 10 μM. E. , F. Neutrophils were treated with zymosan for indicated time periods, followed by measurement of NADPH oxidase activity (E) or Western blot assay using the membrane and cytosol fractions of cell lysates (F). Gas and β-actin were used as inner controls for membrane and cytosolic fractions, respectively. G. Neutrophils were treated with zymosan for 12 h in the presense of NAC (50 mM), SB202190 (10 mM) or NADPH oxidase inhibitor DPI (10 mM) where indicated, followed by measurement of NO and ONOO − production. Data are represented as the mean ± SEM of 3 replicates or representative of 3 independent experiments. * P
    Sb202190, supplied by Biomol GmbH, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Biomol GmbH p38 mapk activation inhibitor
    0.7% ISO reduces ZY-induced <t>p38</t> <t>MAPK</t> activation; p38 MAPK signaling is essential to ZY-induced NF- κ B activation and COX2/PGE 2 generation in vitro . (a) At 0.5 h after ZY or CM (Ctrl) treatment, KCs (1 × 10 6 cells/well in six-well culture plates) were exposed to RA with or without 0.7% ISO for 0.5 h. The cells were continuously stimulated with Ctrl for 0 h and 6 h or ZY (0.5 mg/mL) for 0.5, 1, 2, 3, and 6 h. Western blot analysis was used to assess the phosphorylation of p38 MAPK (Thr180/Tyr182), JNK (Thr183/Tyr185), and ERK1/2 (Thr185/Tyr187). β -Actin was used as the internal control. The ratio from p-p38 MAPK to p38 MAPK is indicated above the bands. (b) The KCs with or without SB202190 (10 μ M) pretreatment for 0.5 h were treated with ZY (0.5 mg/mL) or Ctrl for 1 h. Western blot was performed to determine the phosphorylation of IKK β (Ser180). β -Actin was used as the internal control. The ratio from pIKK β to IKK β is indicated above the bands. (c) The KCs with or without SB202190 (10 μ M) pretreatment for 0.5 h were treated with ZY (0.5 mg/mL) or Ctrl for 24 h. RIA was performed to detect PGE 2 release. Representative data are from three independent experiments and expressed as mean ± SD. * P
    P38 Mapk Activation Inhibitor, supplied by Biomol GmbH, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Isoflurane and zymosan regulate NF-κB activation via ROS/p38 MAPK signaling A. , B. Neutrophils were sequentially treated with zymosan (10 μg/ml) for 15 min, 0.7% isoflurane for 15 min where indicated, and zymosan for 6 h in the presence or absence of NAC (50 mM). Cells were subjected to fluorescence measurement of H 2 O 2 levels (A) and Western blot analysis (B). Scale bar = 10 μM. C. , D. Neutrophils were treated with zymosan for 6 h in the presence of the p38 MAPK inhibitor SB202190 (10 mM) where indicated, followed by immunofluorescent staining of p65 (C) and Western blot analysis of nuclear p65 levels (D). Scale bar = 10 μM. E. , F. Neutrophils were treated with zymosan for indicated time periods, followed by measurement of NADPH oxidase activity (E) or Western blot assay using the membrane and cytosol fractions of cell lysates (F). Gas and β-actin were used as inner controls for membrane and cytosolic fractions, respectively. G. Neutrophils were treated with zymosan for 12 h in the presense of NAC (50 mM), SB202190 (10 mM) or NADPH oxidase inhibitor DPI (10 mM) where indicated, followed by measurement of NO and ONOO − production. Data are represented as the mean ± SEM of 3 replicates or representative of 3 independent experiments. * P

    Journal: Oncotarget

    Article Title: Subanesthetic isoflurane relieves zymosan-induced neutrophil inflammatory response by targeting NMDA glutamate receptor and Toll-like receptor 2 signaling

    doi: 10.18632/oncotarget.9091

    Figure Lengend Snippet: Isoflurane and zymosan regulate NF-κB activation via ROS/p38 MAPK signaling A. , B. Neutrophils were sequentially treated with zymosan (10 μg/ml) for 15 min, 0.7% isoflurane for 15 min where indicated, and zymosan for 6 h in the presence or absence of NAC (50 mM). Cells were subjected to fluorescence measurement of H 2 O 2 levels (A) and Western blot analysis (B). Scale bar = 10 μM. C. , D. Neutrophils were treated with zymosan for 6 h in the presence of the p38 MAPK inhibitor SB202190 (10 mM) where indicated, followed by immunofluorescent staining of p65 (C) and Western blot analysis of nuclear p65 levels (D). Scale bar = 10 μM. E. , F. Neutrophils were treated with zymosan for indicated time periods, followed by measurement of NADPH oxidase activity (E) or Western blot assay using the membrane and cytosol fractions of cell lysates (F). Gas and β-actin were used as inner controls for membrane and cytosolic fractions, respectively. G. Neutrophils were treated with zymosan for 12 h in the presense of NAC (50 mM), SB202190 (10 mM) or NADPH oxidase inhibitor DPI (10 mM) where indicated, followed by measurement of NO and ONOO − production. Data are represented as the mean ± SEM of 3 replicates or representative of 3 independent experiments. * P

    Article Snippet: Diphenyleneiodonium chloride (DPI), N-acetylcysteine (NAC), PP1, and SB202190 were acquired from Biomol (Plymouth Meeting, PA, USA).

    Techniques: Activation Assay, Fluorescence, Western Blot, Staining, Activity Assay

    Effects of garcinol and p300 siRNA on leptin-stimulated A549 cells. Serum-starved A549 cells were pretreated with the OB-R antibody (2 µg/mL), U0126 (10 µM), SB202190 (10 µM), SP600125 (10 µM), Bay11-7082 (10 µM), or garcinol (1 µM) for 1 h. Other cells were transfected with 100 nM p300 siRNA or scrambled siRNA as described in the Experimental section. The cells were then incubated with 1 µg/mL of leptin for the indicated time intervals. At the end of the incubation, the cells were harvested and cell lysates were extracted. ( A , B ) Expression of cPLA 2 -α and p300; ( C ) phosphorylation of p65; and ( D ) acetylation of histone H4 were detected using Western blot with specific antibodies. Cell membranes were stripped and reprobed with the anti-GAPDH antibody as internal controls. The data are expressed as mean ± SEM of 5 independent experiments ( n = 5). p

    Journal: International Journal of Molecular Sciences

    Article Title: Leptin Promotes cPLA2 Gene Expression through Activation of the MAPK/NF-κB/p300 Cascade

    doi: 10.3390/ijms161126045

    Figure Lengend Snippet: Effects of garcinol and p300 siRNA on leptin-stimulated A549 cells. Serum-starved A549 cells were pretreated with the OB-R antibody (2 µg/mL), U0126 (10 µM), SB202190 (10 µM), SP600125 (10 µM), Bay11-7082 (10 µM), or garcinol (1 µM) for 1 h. Other cells were transfected with 100 nM p300 siRNA or scrambled siRNA as described in the Experimental section. The cells were then incubated with 1 µg/mL of leptin for the indicated time intervals. At the end of the incubation, the cells were harvested and cell lysates were extracted. ( A , B ) Expression of cPLA 2 -α and p300; ( C ) phosphorylation of p65; and ( D ) acetylation of histone H4 were detected using Western blot with specific antibodies. Cell membranes were stripped and reprobed with the anti-GAPDH antibody as internal controls. The data are expressed as mean ± SEM of 5 independent experiments ( n = 5). p

    Article Snippet: PD98059, U0126, SB202190, SP600125, and garcinol were obtained from Biomol (Plymouth Meeting, PA, USA).

    Techniques: Transfection, Incubation, Expressing, Western Blot

    Effects of Bay11-7082 on leptin-regulated cPLA 2 -α expression and p65 phosphorylation. Serum-starved A549 cells were pretreated with different concentrations of Bay11-7082, OB-R (2 µg/mL), U0126 (10 µM), SB202190 (10 µM), SP600125 (10 µM), or Bay11-7082 (10 µM) for 1 h. The cells were then stimulated by 1 µg/mL of leptin for the indicated time intervals. At the end of incubation, the cells were harvested and cell lysates were extracted. ( A ) Expression of cPLA 2 -α and ( B ) Ser276 phosphorylation of p65 were detected using Western blot with specific antibodies. The cell membranes were stripped and reprobed with the anti-GAPDH antibody as internal controls. The data are expressed as mean ± SEM of five independent experiments ( n = 5). p

    Journal: International Journal of Molecular Sciences

    Article Title: Leptin Promotes cPLA2 Gene Expression through Activation of the MAPK/NF-κB/p300 Cascade

    doi: 10.3390/ijms161126045

    Figure Lengend Snippet: Effects of Bay11-7082 on leptin-regulated cPLA 2 -α expression and p65 phosphorylation. Serum-starved A549 cells were pretreated with different concentrations of Bay11-7082, OB-R (2 µg/mL), U0126 (10 µM), SB202190 (10 µM), SP600125 (10 µM), or Bay11-7082 (10 µM) for 1 h. The cells were then stimulated by 1 µg/mL of leptin for the indicated time intervals. At the end of incubation, the cells were harvested and cell lysates were extracted. ( A ) Expression of cPLA 2 -α and ( B ) Ser276 phosphorylation of p65 were detected using Western blot with specific antibodies. The cell membranes were stripped and reprobed with the anti-GAPDH antibody as internal controls. The data are expressed as mean ± SEM of five independent experiments ( n = 5). p

    Article Snippet: PD98059, U0126, SB202190, SP600125, and garcinol were obtained from Biomol (Plymouth Meeting, PA, USA).

    Techniques: Expressing, Incubation, Western Blot

    Effects of various inhibitors on leptin-stimulated cPLA 2 -α mRNA expression. Serum-starved A549 cells were pretreated with OB-R (2 µg/mL), PD98059 (10 µM), U0126 (10 µM), SB202190 (10 µM), SP600125 (10 µM), Bay11-7082 (10 µM), or garcinol (1 µM) for 1 h. The cells were then incubated with 1 µg/mL of leptin for 6 h. At the end of incubation, mRNA were extracted and used as templates of cDNA. Expression of cPLA 2 -α mRNA was detected using RT-PCR. Data are expressed as mean ± SEM of five independent experiments ( n = 5). p

    Journal: International Journal of Molecular Sciences

    Article Title: Leptin Promotes cPLA2 Gene Expression through Activation of the MAPK/NF-κB/p300 Cascade

    doi: 10.3390/ijms161126045

    Figure Lengend Snippet: Effects of various inhibitors on leptin-stimulated cPLA 2 -α mRNA expression. Serum-starved A549 cells were pretreated with OB-R (2 µg/mL), PD98059 (10 µM), U0126 (10 µM), SB202190 (10 µM), SP600125 (10 µM), Bay11-7082 (10 µM), or garcinol (1 µM) for 1 h. The cells were then incubated with 1 µg/mL of leptin for 6 h. At the end of incubation, mRNA were extracted and used as templates of cDNA. Expression of cPLA 2 -α mRNA was detected using RT-PCR. Data are expressed as mean ± SEM of five independent experiments ( n = 5). p

    Article Snippet: PD98059, U0126, SB202190, SP600125, and garcinol were obtained from Biomol (Plymouth Meeting, PA, USA).

    Techniques: Expressing, Incubation, Reverse Transcription Polymerase Chain Reaction

    SB202190 attenuation of cPLA 2 -α expression and p38 MAPK phosphorylation. Serum-starved A549 cells were pretreated with different concentrations of SB202190 or the OB-R antibody for 1 h and then stimulated by 1 µg/mL of leptin for the indicated time intervals. At the end of incubation, the cells were harvested and cell lysates were extracted. ( A ) Expression of cPLA 2 -α and ( B , C ) phosphorylation of p38 MAPK were detected using Western blot with specific antibodies. Cell membranes were stripped and reprobed with the anti-GAPDH antibody as internal controls. The data are expressed as mean ± SEM of five independent experiments ( n = 5). p

    Journal: International Journal of Molecular Sciences

    Article Title: Leptin Promotes cPLA2 Gene Expression through Activation of the MAPK/NF-κB/p300 Cascade

    doi: 10.3390/ijms161126045

    Figure Lengend Snippet: SB202190 attenuation of cPLA 2 -α expression and p38 MAPK phosphorylation. Serum-starved A549 cells were pretreated with different concentrations of SB202190 or the OB-R antibody for 1 h and then stimulated by 1 µg/mL of leptin for the indicated time intervals. At the end of incubation, the cells were harvested and cell lysates were extracted. ( A ) Expression of cPLA 2 -α and ( B , C ) phosphorylation of p38 MAPK were detected using Western blot with specific antibodies. Cell membranes were stripped and reprobed with the anti-GAPDH antibody as internal controls. The data are expressed as mean ± SEM of five independent experiments ( n = 5). p

    Article Snippet: PD98059, U0126, SB202190, SP600125, and garcinol were obtained from Biomol (Plymouth Meeting, PA, USA).

    Techniques: Expressing, Incubation, Western Blot

    Involvement of PKC α -dependent p38 MAPK and JNK1/2 in TNF- α -mediated ROS generation and cPLA 2 expression. (a) Cells were pretreated with U0126, SB202190, or SP600125, or combinatorial treatment for 1 h, and then incubated with TNF- α for 16 h. The expression of cPLA 2 was determined by Western blotting. (b) Cells were pretreated with or without U0126, SB202190, or SP600125 for 1 h and then incubated with TNF- α for the indicated time intervals. (c) Cells were transfected with scrambled, p38, or JNK1 siRNA and then incubated with TNF- α for 16 h. The levels of p38, JNK1, and cPLA 2 were determined by Western blotting. (d) Cells were pretreated with Gö6976 for 1 h and then stimulated with TNF- α for 30 min. The levels of phospho-p38 MAPK and phospho-JNK1/2 were determined by Western blotting. (e) Cells were pretreated with NAC for 1 h, and then incubated with TNF- α for the indicated time intervals. The levels of phospho-p38 MAPK and phospho-JNK1/2 were determined by Western blotting. (f) Cells were pretreated with SB202190 (1 μ M) or SP600125 (1 μ M) for 1 h before exposure to TNF- α for 1 h (shaded bars) or 2 h (open bars). The NOX activity (shaded bars) and ROS generation (open bars) were analyzed. (g) Cells were pretreated with SB202190 (1 μ M) or SP600125 (1 μ M) for 1 h before exposure to TNF- α for 1 h. The membrane (ME) and cytosolic (CE) fractions were prepared and subjected to Western blotting using an anti-p47 phox antibody. All analyses were performed on samples from 4 RA patients. Results are representative of 3 independent experiments. Values in (a), (c), (f), and (g) are the mean ± SEM. * P

    Journal: Mediators of Inflammation

    Article Title: HO-1 Induction by CO-RM2 Attenuates TNF-α-Induced Cytosolic Phospholipase A2 Expression via Inhibition of PKCα-Dependent NADPH Oxidase/ROS and NF-κB

    doi: 10.1155/2014/279171

    Figure Lengend Snippet: Involvement of PKC α -dependent p38 MAPK and JNK1/2 in TNF- α -mediated ROS generation and cPLA 2 expression. (a) Cells were pretreated with U0126, SB202190, or SP600125, or combinatorial treatment for 1 h, and then incubated with TNF- α for 16 h. The expression of cPLA 2 was determined by Western blotting. (b) Cells were pretreated with or without U0126, SB202190, or SP600125 for 1 h and then incubated with TNF- α for the indicated time intervals. (c) Cells were transfected with scrambled, p38, or JNK1 siRNA and then incubated with TNF- α for 16 h. The levels of p38, JNK1, and cPLA 2 were determined by Western blotting. (d) Cells were pretreated with Gö6976 for 1 h and then stimulated with TNF- α for 30 min. The levels of phospho-p38 MAPK and phospho-JNK1/2 were determined by Western blotting. (e) Cells were pretreated with NAC for 1 h, and then incubated with TNF- α for the indicated time intervals. The levels of phospho-p38 MAPK and phospho-JNK1/2 were determined by Western blotting. (f) Cells were pretreated with SB202190 (1 μ M) or SP600125 (1 μ M) for 1 h before exposure to TNF- α for 1 h (shaded bars) or 2 h (open bars). The NOX activity (shaded bars) and ROS generation (open bars) were analyzed. (g) Cells were pretreated with SB202190 (1 μ M) or SP600125 (1 μ M) for 1 h before exposure to TNF- α for 1 h. The membrane (ME) and cytosolic (CE) fractions were prepared and subjected to Western blotting using an anti-p47 phox antibody. All analyses were performed on samples from 4 RA patients. Results are representative of 3 independent experiments. Values in (a), (c), (f), and (g) are the mean ± SEM. * P

    Article Snippet: Materials Diphenyleneiodonium chloride (DPI), Gö6976, U0126, SB202190, SP600125, and helenalin were obtained from Biomol (Plymouth Meeting, PA).

    Techniques: Expressing, Incubation, Western Blot, Transfection, Activity Assay

    TNF- α -mediated ROS-dependent activation of NF- κ B is required for TNF- α -induced cPLA 2 expression. (a) RASFs were pretreated with helenalin for 1 h and then incubated with TNF- α for 16 h. The expression of cPLA 2 was determined by Western blotting. (b) Cells were transfected with scrambled or p65 siRNA and then incubated with TNF- α for 16 h. The levels of p65 and cPLA 2 protein were determined by Western blotting. (c) Cells were pretreated with or without Gö6976 (1 μ M), SB202190 (1 μ M), SP600125 (1 μ M), or NAC (10 mM) for 1 h before exposure to TNF- α for the indicated time intervals. The levels of phospho-IKK α / β and phospho-p65 were determined by Western blotting. Cells were pretreated with Gö6976 (1 μ M), SB201290 (1 μ M), SP600125 (1 μ M), NAC (10 mM), or helenalin (1 μ M) for 1 h and then incubated with TNF- α for 30 min. (d) Cells were fixed and then labeled using an anti-p65 antibody and FITC-conjugated secondary antibody. (e) The nuclear extract (NE) was prepared and subjected to Western blotting using the indicated antibodies and lamin A used as an internal control. (f) Cells were transiently transfected with NF- κ B-Luc reporter gene, pretreated with Gö6976, SB201290, SP600125, DPI, APO, NAC, or helenalin for 1 h, and then incubated with TNF- α for 4 h. The promoter activity was determined. (g) Cells were pretreated with Gö6976, SB201290, SP600125, DPI, or NAC for 1 h and incubated with TNF- α for 2 h. Chromatin was immunoprecipitated using an anti-p65 antibody. The associated cPLA 2 promoter DNA was amplified by polymerase chain reaction (PCR). Input represents PCR products from chromatin pellets before immunoprecipitation. (h) Cells were transfected without or with a cPLA 2 -Luc reporter gene, pretreated with Gö6976, SB201290, SP600125, DPI, APO, NAC, or helenalin for 1 h, and then incubated with TNF- α for 6 h. The mRNA levels of cPLA 2 (open bars) and cPLA 2 -luc promoter (shaded bars) were measured. (i) The PGE 2 levels in the media were analyzed using a PGE 2 EIA kit. All analyses were performed on samples from 4 RA patients. Results are representative of 3 independent experiments. Values in (a), (b), (e), (f), and (g) are the mean ± SEM. * P

    Journal: Mediators of Inflammation

    Article Title: HO-1 Induction by CO-RM2 Attenuates TNF-α-Induced Cytosolic Phospholipase A2 Expression via Inhibition of PKCα-Dependent NADPH Oxidase/ROS and NF-κB

    doi: 10.1155/2014/279171

    Figure Lengend Snippet: TNF- α -mediated ROS-dependent activation of NF- κ B is required for TNF- α -induced cPLA 2 expression. (a) RASFs were pretreated with helenalin for 1 h and then incubated with TNF- α for 16 h. The expression of cPLA 2 was determined by Western blotting. (b) Cells were transfected with scrambled or p65 siRNA and then incubated with TNF- α for 16 h. The levels of p65 and cPLA 2 protein were determined by Western blotting. (c) Cells were pretreated with or without Gö6976 (1 μ M), SB202190 (1 μ M), SP600125 (1 μ M), or NAC (10 mM) for 1 h before exposure to TNF- α for the indicated time intervals. The levels of phospho-IKK α / β and phospho-p65 were determined by Western blotting. Cells were pretreated with Gö6976 (1 μ M), SB201290 (1 μ M), SP600125 (1 μ M), NAC (10 mM), or helenalin (1 μ M) for 1 h and then incubated with TNF- α for 30 min. (d) Cells were fixed and then labeled using an anti-p65 antibody and FITC-conjugated secondary antibody. (e) The nuclear extract (NE) was prepared and subjected to Western blotting using the indicated antibodies and lamin A used as an internal control. (f) Cells were transiently transfected with NF- κ B-Luc reporter gene, pretreated with Gö6976, SB201290, SP600125, DPI, APO, NAC, or helenalin for 1 h, and then incubated with TNF- α for 4 h. The promoter activity was determined. (g) Cells were pretreated with Gö6976, SB201290, SP600125, DPI, or NAC for 1 h and incubated with TNF- α for 2 h. Chromatin was immunoprecipitated using an anti-p65 antibody. The associated cPLA 2 promoter DNA was amplified by polymerase chain reaction (PCR). Input represents PCR products from chromatin pellets before immunoprecipitation. (h) Cells were transfected without or with a cPLA 2 -Luc reporter gene, pretreated with Gö6976, SB201290, SP600125, DPI, APO, NAC, or helenalin for 1 h, and then incubated with TNF- α for 6 h. The mRNA levels of cPLA 2 (open bars) and cPLA 2 -luc promoter (shaded bars) were measured. (i) The PGE 2 levels in the media were analyzed using a PGE 2 EIA kit. All analyses were performed on samples from 4 RA patients. Results are representative of 3 independent experiments. Values in (a), (b), (e), (f), and (g) are the mean ± SEM. * P

    Article Snippet: Materials Diphenyleneiodonium chloride (DPI), Gö6976, U0126, SB202190, SP600125, and helenalin were obtained from Biomol (Plymouth Meeting, PA).

    Techniques: Activation Assay, Expressing, Incubation, Western Blot, Transfection, Labeling, Activity Assay, Immunoprecipitation, Amplification, Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay

    0.7% ISO reduces ZY-induced p38 MAPK activation; p38 MAPK signaling is essential to ZY-induced NF- κ B activation and COX2/PGE 2 generation in vitro . (a) At 0.5 h after ZY or CM (Ctrl) treatment, KCs (1 × 10 6 cells/well in six-well culture plates) were exposed to RA with or without 0.7% ISO for 0.5 h. The cells were continuously stimulated with Ctrl for 0 h and 6 h or ZY (0.5 mg/mL) for 0.5, 1, 2, 3, and 6 h. Western blot analysis was used to assess the phosphorylation of p38 MAPK (Thr180/Tyr182), JNK (Thr183/Tyr185), and ERK1/2 (Thr185/Tyr187). β -Actin was used as the internal control. The ratio from p-p38 MAPK to p38 MAPK is indicated above the bands. (b) The KCs with or without SB202190 (10 μ M) pretreatment for 0.5 h were treated with ZY (0.5 mg/mL) or Ctrl for 1 h. Western blot was performed to determine the phosphorylation of IKK β (Ser180). β -Actin was used as the internal control. The ratio from pIKK β to IKK β is indicated above the bands. (c) The KCs with or without SB202190 (10 μ M) pretreatment for 0.5 h were treated with ZY (0.5 mg/mL) or Ctrl for 24 h. RIA was performed to detect PGE 2 release. Representative data are from three independent experiments and expressed as mean ± SD. * P

    Journal: Oxidative Medicine and Cellular Longevity

    Article Title: Subanesthetic Isoflurane Reduces Zymosan-Induced Inflammation in Murine Kupffer Cells by Inhibiting ROS-Activated p38 MAPK/NF-κB Signaling

    doi: 10.1155/2014/851692

    Figure Lengend Snippet: 0.7% ISO reduces ZY-induced p38 MAPK activation; p38 MAPK signaling is essential to ZY-induced NF- κ B activation and COX2/PGE 2 generation in vitro . (a) At 0.5 h after ZY or CM (Ctrl) treatment, KCs (1 × 10 6 cells/well in six-well culture plates) were exposed to RA with or without 0.7% ISO for 0.5 h. The cells were continuously stimulated with Ctrl for 0 h and 6 h or ZY (0.5 mg/mL) for 0.5, 1, 2, 3, and 6 h. Western blot analysis was used to assess the phosphorylation of p38 MAPK (Thr180/Tyr182), JNK (Thr183/Tyr185), and ERK1/2 (Thr185/Tyr187). β -Actin was used as the internal control. The ratio from p-p38 MAPK to p38 MAPK is indicated above the bands. (b) The KCs with or without SB202190 (10 μ M) pretreatment for 0.5 h were treated with ZY (0.5 mg/mL) or Ctrl for 1 h. Western blot was performed to determine the phosphorylation of IKK β (Ser180). β -Actin was used as the internal control. The ratio from pIKK β to IKK β is indicated above the bands. (c) The KCs with or without SB202190 (10 μ M) pretreatment for 0.5 h were treated with ZY (0.5 mg/mL) or Ctrl for 24 h. RIA was performed to detect PGE 2 release. Representative data are from three independent experiments and expressed as mean ± SD. * P

    Article Snippet: ROS scavenger (Edaravone, MCI-186) was from Tocris Bioscience (Ellisville, MO, USA). p38 MAPK activation inhibitor (SB202190) and NF-κ B activation inhibitor (NAI) were purchased from Biomol (Plymouth Meeting, PA, USA) and Calbiochem (Darmstadt, Germany), respectively.

    Techniques: Activation Assay, In Vitro, Western Blot

    0.7% ISO reduces ZY-induced ROS generation; ROS is essential to ZY-induced activation of p38 MAPK and NF- κ B, as well as PGE 2 production. (a) At 0.5 h after KCs (1 × 10 6 cells/well in six-well culture plates) were treated with ZY or CM (Ctrl) for 0.5 h with or without MCI-186 (50 μ M) pretreatment for 0.5 h, the cells were exposed to RA with or without 0.7% ISO for another 0.5 h. The KCs were continuously treated with ZY (0.5 mg/mL) or Ctrl for 24 h. RIA was performed to detect PGE 2 production. (b) The KCs with or without MCI-186 (50 μ M) pretreatment for 0.5 h were treated with ZY (0.5 mg/mL) or Ctrl for 1 h. Western blot was performed to determine the phosphorylation of p38 MAPK (Thr180/Tyr182) and NF- κ B (Ser536). β -Actin and lamin B were used as the internal controls. The ratios from p-p38 MAPK to p38 MAPK and from pNF- κ B to NF- κ B are indicated above the bands. (c) The KCs (5 × 10 4 cells/well in 96-well culture plates) with or without MCI-186 (50 μ M) pretreatment for 0.5 h were stimulated with ZY (0.5 mg/mL) for 1 h with or without 0.7% ISO posttreatment for 0.5 h. DCFH-DA was used to assess the production of intracellular ROS. Representative data are from three independent experiments and expressed as mean ± SD. * P

    Journal: Oxidative Medicine and Cellular Longevity

    Article Title: Subanesthetic Isoflurane Reduces Zymosan-Induced Inflammation in Murine Kupffer Cells by Inhibiting ROS-Activated p38 MAPK/NF-κB Signaling

    doi: 10.1155/2014/851692

    Figure Lengend Snippet: 0.7% ISO reduces ZY-induced ROS generation; ROS is essential to ZY-induced activation of p38 MAPK and NF- κ B, as well as PGE 2 production. (a) At 0.5 h after KCs (1 × 10 6 cells/well in six-well culture plates) were treated with ZY or CM (Ctrl) for 0.5 h with or without MCI-186 (50 μ M) pretreatment for 0.5 h, the cells were exposed to RA with or without 0.7% ISO for another 0.5 h. The KCs were continuously treated with ZY (0.5 mg/mL) or Ctrl for 24 h. RIA was performed to detect PGE 2 production. (b) The KCs with or without MCI-186 (50 μ M) pretreatment for 0.5 h were treated with ZY (0.5 mg/mL) or Ctrl for 1 h. Western blot was performed to determine the phosphorylation of p38 MAPK (Thr180/Tyr182) and NF- κ B (Ser536). β -Actin and lamin B were used as the internal controls. The ratios from p-p38 MAPK to p38 MAPK and from pNF- κ B to NF- κ B are indicated above the bands. (c) The KCs (5 × 10 4 cells/well in 96-well culture plates) with or without MCI-186 (50 μ M) pretreatment for 0.5 h were stimulated with ZY (0.5 mg/mL) for 1 h with or without 0.7% ISO posttreatment for 0.5 h. DCFH-DA was used to assess the production of intracellular ROS. Representative data are from three independent experiments and expressed as mean ± SD. * P

    Article Snippet: ROS scavenger (Edaravone, MCI-186) was from Tocris Bioscience (Ellisville, MO, USA). p38 MAPK activation inhibitor (SB202190) and NF-κ B activation inhibitor (NAI) were purchased from Biomol (Plymouth Meeting, PA, USA) and Calbiochem (Darmstadt, Germany), respectively.

    Techniques: Activation Assay, Western Blot