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Merck KGaA sb202190
( a ) Inhibition of <t>SB202190</t> on cell viability: MTT assays in HepG2, BEL7404 and HL7702 cells treated with SB202190 for 48 h at different concentrations (0, 2.5, 5, 10, 25 and 50 μM); ( b ) Western blot: Displaying that SB202190 dose-dependently inhibits the phosphorylation of p38 downstream proteins. HepG2 cells were treated with SB202190 for 24 h at different concentrations (0, 10, 25 and 50 μM); ( c – g ) HepG2 cells were treated with 25 μM SB202190 at 24 h after transfecting with pcDNA3.1(−)-Pokemon or pcDNA3.1(−): ( c ) HepG2 Cell growth rate; ( d ) Effect of Pokemon and p38 inhibitor SB202190 on colony formation in HepG2 cells, the colony formation rate stands for the proportion of final clone number accounted for in plated cell number; ( e ) In vitro migration assays; ( f ) In vitro invasion assays. Bar chart below the photo stands for the relative fold of the migrated or invaded cell number compared to the negative control group; ( g ) Pokemon activates p38 signaling pathway in hepatic cells: Left panel is Western blot bands. Western blot in HepG2 cells after Pokemon was overexpressed for 60 h, and the cells were treated by SB202190 at the concentration of 25 μM; right panel is quantification of western blot data. * p
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1) Product Images from "p38?, A Novel Regulatory Target of Pokemon in Hepatic Cells"

Article Title: p38?, A Novel Regulatory Target of Pokemon in Hepatic Cells

Journal: International Journal of Molecular Sciences

doi: 10.3390/ijms140713511

( a ) Inhibition of SB202190 on cell viability: MTT assays in HepG2, BEL7404 and HL7702 cells treated with SB202190 for 48 h at different concentrations (0, 2.5, 5, 10, 25 and 50 μM); ( b ) Western blot: Displaying that SB202190 dose-dependently inhibits the phosphorylation of p38 downstream proteins. HepG2 cells were treated with SB202190 for 24 h at different concentrations (0, 10, 25 and 50 μM); ( c – g ) HepG2 cells were treated with 25 μM SB202190 at 24 h after transfecting with pcDNA3.1(−)-Pokemon or pcDNA3.1(−): ( c ) HepG2 Cell growth rate; ( d ) Effect of Pokemon and p38 inhibitor SB202190 on colony formation in HepG2 cells, the colony formation rate stands for the proportion of final clone number accounted for in plated cell number; ( e ) In vitro migration assays; ( f ) In vitro invasion assays. Bar chart below the photo stands for the relative fold of the migrated or invaded cell number compared to the negative control group; ( g ) Pokemon activates p38 signaling pathway in hepatic cells: Left panel is Western blot bands. Western blot in HepG2 cells after Pokemon was overexpressed for 60 h, and the cells were treated by SB202190 at the concentration of 25 μM; right panel is quantification of western blot data. * p
Figure Legend Snippet: ( a ) Inhibition of SB202190 on cell viability: MTT assays in HepG2, BEL7404 and HL7702 cells treated with SB202190 for 48 h at different concentrations (0, 2.5, 5, 10, 25 and 50 μM); ( b ) Western blot: Displaying that SB202190 dose-dependently inhibits the phosphorylation of p38 downstream proteins. HepG2 cells were treated with SB202190 for 24 h at different concentrations (0, 10, 25 and 50 μM); ( c – g ) HepG2 cells were treated with 25 μM SB202190 at 24 h after transfecting with pcDNA3.1(−)-Pokemon or pcDNA3.1(−): ( c ) HepG2 Cell growth rate; ( d ) Effect of Pokemon and p38 inhibitor SB202190 on colony formation in HepG2 cells, the colony formation rate stands for the proportion of final clone number accounted for in plated cell number; ( e ) In vitro migration assays; ( f ) In vitro invasion assays. Bar chart below the photo stands for the relative fold of the migrated or invaded cell number compared to the negative control group; ( g ) Pokemon activates p38 signaling pathway in hepatic cells: Left panel is Western blot bands. Western blot in HepG2 cells after Pokemon was overexpressed for 60 h, and the cells were treated by SB202190 at the concentration of 25 μM; right panel is quantification of western blot data. * p

Techniques Used: Inhibition, MTT Assay, Western Blot, In Vitro, Migration, Negative Control, Concentration Assay

2) Product Images from "Transcription of Tnfaip3 Is Regulated by NF-?B and p38 via C/EBP? in Activated Macrophages"

Article Title: Transcription of Tnfaip3 Is Regulated by NF-?B and p38 via C/EBP? in Activated Macrophages

Journal: PLoS ONE

doi: 10.1371/journal.pone.0073153

NF-κB and C/EBPβ are required for induction of Tnfaip3 in LPS-activated macrophages. (A) Schematic map of predicted NF-κB p65 and C/EBPβ DNA binding sites in the promoter of Tnfaip3 . oPOSSUM ( http://www.cisreg.ca/oPOSSUM/ ) was used as the prediction tool. The JASPAR CORE vertebrate database was selected for transcription factor binding site matrices. Two arrows depict the PCR primers. (B) p65 and C/EBPβ bind to the promoter of Tnfaip3 after LPS stimulation. RAW264.7 cells were treated with LPS (100 ng/ml) for the indicated times. Chromatin was immunoprecipitated with anti-p65 and anti-C/EBPβ antibodies. Rabbit IgG was a negative control. Precipitated DNA or 1% of the chromatin input was amplified with primers for the Tnfaip3 promoter (−89 ∼ −410). The PCR products were loaded and separated on a 2% agarose gel. One of two independent experiments is shown. (C) LPS-induced association of p65 and C/EBPβ with Tnfaip3 was reduced in the presence of p38 inhibition. Chromatin isolated from RAW264.7 cells treated with LPS (100 ng/ml) for 4 h in the absence or presence of SB202190 (10 µM) were subjected to ChIP assay as described above. The relative quantity of promoter enriched by ChIP was quantified by real-time PCR and expressed as the fold enrichment of untreated control samples after normalization to rabbit IgG. Data represent the means of two independent experiments. (D) LPS-induced expression of A20 (TNFAIP3) was decreased in C/EBPβ-depleted RAW264.7 cells. Cells were infected with lentiviruses encoding shRNA against luciferase ( shLuc ) or C/EBPβ ( shCebpb ), and treated with LPS (100 ng/ml) for the indicated times. Cell lysates were collected and analyzed by immunoblotting using the indicated antibodies.
Figure Legend Snippet: NF-κB and C/EBPβ are required for induction of Tnfaip3 in LPS-activated macrophages. (A) Schematic map of predicted NF-κB p65 and C/EBPβ DNA binding sites in the promoter of Tnfaip3 . oPOSSUM ( http://www.cisreg.ca/oPOSSUM/ ) was used as the prediction tool. The JASPAR CORE vertebrate database was selected for transcription factor binding site matrices. Two arrows depict the PCR primers. (B) p65 and C/EBPβ bind to the promoter of Tnfaip3 after LPS stimulation. RAW264.7 cells were treated with LPS (100 ng/ml) for the indicated times. Chromatin was immunoprecipitated with anti-p65 and anti-C/EBPβ antibodies. Rabbit IgG was a negative control. Precipitated DNA or 1% of the chromatin input was amplified with primers for the Tnfaip3 promoter (−89 ∼ −410). The PCR products were loaded and separated on a 2% agarose gel. One of two independent experiments is shown. (C) LPS-induced association of p65 and C/EBPβ with Tnfaip3 was reduced in the presence of p38 inhibition. Chromatin isolated from RAW264.7 cells treated with LPS (100 ng/ml) for 4 h in the absence or presence of SB202190 (10 µM) were subjected to ChIP assay as described above. The relative quantity of promoter enriched by ChIP was quantified by real-time PCR and expressed as the fold enrichment of untreated control samples after normalization to rabbit IgG. Data represent the means of two independent experiments. (D) LPS-induced expression of A20 (TNFAIP3) was decreased in C/EBPβ-depleted RAW264.7 cells. Cells were infected with lentiviruses encoding shRNA against luciferase ( shLuc ) or C/EBPβ ( shCebpb ), and treated with LPS (100 ng/ml) for the indicated times. Cell lysates were collected and analyzed by immunoblotting using the indicated antibodies.

Techniques Used: Binding Assay, Polymerase Chain Reaction, Immunoprecipitation, Negative Control, Amplification, Agarose Gel Electrophoresis, Inhibition, Isolation, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Expressing, Infection, shRNA, Luciferase

Depletion of IKKβ expression and inhibition of p38 signaling pathway in Ikkβ Δ and SB202190-treated bone marrow-derived macrophages (BMDMs). (A) Immunoblotting of IKKβ and p38 from BMDMs isolated from wild-type (wt; Ikkβ F/F ) and Ikkβ Δ mice. (B) Immunoblotting of p38 and phosphorylated p38 from wt BMDMs treated with LPS (100 ng/mL) in the absence or presence of SB202190 (10 µM) for 2 h. (C D) mRNA expression levels of IL-1β and IL-6 were inhibited in SB202190-treated BMDMs after LPS treatment. The expression levels of Il1b (C) and Il6 (D) were determined from wt BMDMs treated with LPS (100 ng/mL) for 4 h in the absence or presence of SB202190 (10 µM) for 2 h using real-time RT-PCR. Data represent the mean ± SEM for three independent experiments. ***, P
Figure Legend Snippet: Depletion of IKKβ expression and inhibition of p38 signaling pathway in Ikkβ Δ and SB202190-treated bone marrow-derived macrophages (BMDMs). (A) Immunoblotting of IKKβ and p38 from BMDMs isolated from wild-type (wt; Ikkβ F/F ) and Ikkβ Δ mice. (B) Immunoblotting of p38 and phosphorylated p38 from wt BMDMs treated with LPS (100 ng/mL) in the absence or presence of SB202190 (10 µM) for 2 h. (C D) mRNA expression levels of IL-1β and IL-6 were inhibited in SB202190-treated BMDMs after LPS treatment. The expression levels of Il1b (C) and Il6 (D) were determined from wt BMDMs treated with LPS (100 ng/mL) for 4 h in the absence or presence of SB202190 (10 µM) for 2 h using real-time RT-PCR. Data represent the mean ± SEM for three independent experiments. ***, P

Techniques Used: Expressing, Inhibition, Derivative Assay, Isolation, Mouse Assay, Quantitative RT-PCR

Identification of LPS-induced genes that were regulated by both NF-κB and p38. (A) Venn diagram of NF-κB and p38-dependent genes. NF-κB-related genes were identified from genes that were down-regulated in Ikkβ Δ BMDMs as compared with wt BMDMs after LPS treatment, and p38-related genes were selected by comparing SB202190- (p38-inhibitor) with dimethyl sulfoxide (DMSO)-treated BMDMs after LPS treatment. Thirty-two LPS-induced genes were regulated by both NF-κB and p38-downstream transcription factors. (B) Hierarchical clustering of average fold change for the NF-κB and p38-dependent genes. Each column represents the average fold change 4 h after LPS treatment compared to 0 h. *: genes chosen for PCR validation. (C) Relative fold changes of Il1b, Serpinb2, Tnfaip3 , and Zc3h12a mRNA from BMDMs from wt and Ikkβ Δ cells stimulated with LPS (100 ng/mL) for 4 h in the presence or absence of SB202190 (10 µM) were measured by quantitative RT-PCR. The fold change of 4 h vs. 0 h was normalized to wt BMDMs. The internal control was Cyclophilin A mRNA ( Cypa ). Data represent the mean ± SD for at least two independent experiments. *, P
Figure Legend Snippet: Identification of LPS-induced genes that were regulated by both NF-κB and p38. (A) Venn diagram of NF-κB and p38-dependent genes. NF-κB-related genes were identified from genes that were down-regulated in Ikkβ Δ BMDMs as compared with wt BMDMs after LPS treatment, and p38-related genes were selected by comparing SB202190- (p38-inhibitor) with dimethyl sulfoxide (DMSO)-treated BMDMs after LPS treatment. Thirty-two LPS-induced genes were regulated by both NF-κB and p38-downstream transcription factors. (B) Hierarchical clustering of average fold change for the NF-κB and p38-dependent genes. Each column represents the average fold change 4 h after LPS treatment compared to 0 h. *: genes chosen for PCR validation. (C) Relative fold changes of Il1b, Serpinb2, Tnfaip3 , and Zc3h12a mRNA from BMDMs from wt and Ikkβ Δ cells stimulated with LPS (100 ng/mL) for 4 h in the presence or absence of SB202190 (10 µM) were measured by quantitative RT-PCR. The fold change of 4 h vs. 0 h was normalized to wt BMDMs. The internal control was Cyclophilin A mRNA ( Cypa ). Data represent the mean ± SD for at least two independent experiments. *, P

Techniques Used: Polymerase Chain Reaction, Quantitative RT-PCR

C/EBPβ and A20 (TNFAIP3) were suppressed in Ikkβ Δ and p38-inhibited macrophages. (A–C) Expression levels of Tnfaip3 and Cebpb mRNA were decreased in Ikkβ Δ and p38-inhibited BMDMs in response to LPS. BMDMs from wt and Ikkβ Δ cells treated with or without SB202190 (10 µM) were stimulated with LPS (100 ng/mL) for 4 h. Total RNAs were isolated and analyzed by semi-quantitative RT-PCR (A) or quantitative real-time RT-PCR for expression of Cebpb (B) and Tnfaip3 (C) mRNAs. Results were normalized to Cyclophilin A ( Cypa ) and are presented relative to expression in wt BMDMs. * P
Figure Legend Snippet: C/EBPβ and A20 (TNFAIP3) were suppressed in Ikkβ Δ and p38-inhibited macrophages. (A–C) Expression levels of Tnfaip3 and Cebpb mRNA were decreased in Ikkβ Δ and p38-inhibited BMDMs in response to LPS. BMDMs from wt and Ikkβ Δ cells treated with or without SB202190 (10 µM) were stimulated with LPS (100 ng/mL) for 4 h. Total RNAs were isolated and analyzed by semi-quantitative RT-PCR (A) or quantitative real-time RT-PCR for expression of Cebpb (B) and Tnfaip3 (C) mRNAs. Results were normalized to Cyclophilin A ( Cypa ) and are presented relative to expression in wt BMDMs. * P

Techniques Used: Expressing, Isolation, Quantitative RT-PCR

3) Product Images from "p38?, A Novel Regulatory Target of Pokemon in Hepatic Cells"

Article Title: p38?, A Novel Regulatory Target of Pokemon in Hepatic Cells

Journal: International Journal of Molecular Sciences

doi: 10.3390/ijms140713511

( a ) Inhibition of SB202190 on cell viability: MTT assays in HepG2, BEL7404 and HL7702 cells treated with SB202190 for 48 h at different concentrations (0, 2.5, 5, 10, 25 and 50 μM); ( b ) Western blot: Displaying that SB202190 dose-dependently inhibits the phosphorylation of p38 downstream proteins. HepG2 cells were treated with SB202190 for 24 h at different concentrations (0, 10, 25 and 50 μM); ( c – g ) HepG2 cells were treated with 25 μM SB202190 at 24 h after transfecting with pcDNA3.1(−)-Pokemon or pcDNA3.1(−): ( c ) HepG2 Cell growth rate; ( d ) Effect of Pokemon and p38 inhibitor SB202190 on colony formation in HepG2 cells, the colony formation rate stands for the proportion of final clone number accounted for in plated cell number; ( e ) In vitro migration assays; ( f ) In vitro invasion assays. Bar chart below the photo stands for the relative fold of the migrated or invaded cell number compared to the negative control group; ( g ) Pokemon activates p38 signaling pathway in hepatic cells: Left panel is Western blot bands. Western blot in HepG2 cells after Pokemon was overexpressed for 60 h, and the cells were treated by SB202190 at the concentration of 25 μM; right panel is quantification of western blot data. * p
Figure Legend Snippet: ( a ) Inhibition of SB202190 on cell viability: MTT assays in HepG2, BEL7404 and HL7702 cells treated with SB202190 for 48 h at different concentrations (0, 2.5, 5, 10, 25 and 50 μM); ( b ) Western blot: Displaying that SB202190 dose-dependently inhibits the phosphorylation of p38 downstream proteins. HepG2 cells were treated with SB202190 for 24 h at different concentrations (0, 10, 25 and 50 μM); ( c – g ) HepG2 cells were treated with 25 μM SB202190 at 24 h after transfecting with pcDNA3.1(−)-Pokemon or pcDNA3.1(−): ( c ) HepG2 Cell growth rate; ( d ) Effect of Pokemon and p38 inhibitor SB202190 on colony formation in HepG2 cells, the colony formation rate stands for the proportion of final clone number accounted for in plated cell number; ( e ) In vitro migration assays; ( f ) In vitro invasion assays. Bar chart below the photo stands for the relative fold of the migrated or invaded cell number compared to the negative control group; ( g ) Pokemon activates p38 signaling pathway in hepatic cells: Left panel is Western blot bands. Western blot in HepG2 cells after Pokemon was overexpressed for 60 h, and the cells were treated by SB202190 at the concentration of 25 μM; right panel is quantification of western blot data. * p

Techniques Used: Inhibition, MTT Assay, Western Blot, In Vitro, Migration, Negative Control, Concentration Assay

4) Product Images from "Transcription of Tnfaip3 Is Regulated by NF-?B and p38 via C/EBP? in Activated Macrophages"

Article Title: Transcription of Tnfaip3 Is Regulated by NF-?B and p38 via C/EBP? in Activated Macrophages

Journal: PLoS ONE

doi: 10.1371/journal.pone.0073153

NF-κB and C/EBPβ are required for induction of Tnfaip3 in LPS-activated macrophages. (A) Schematic map of predicted NF-κB p65 and C/EBPβ DNA binding sites in the promoter of Tnfaip3 . oPOSSUM ( http://www.cisreg.ca/oPOSSUM/ ) was used as the prediction tool. The JASPAR CORE vertebrate database was selected for transcription factor binding site matrices. Two arrows depict the PCR primers. (B) p65 and C/EBPβ bind to the promoter of Tnfaip3 after LPS stimulation. RAW264.7 cells were treated with LPS (100 ng/ml) for the indicated times. Chromatin was immunoprecipitated with anti-p65 and anti-C/EBPβ antibodies. Rabbit IgG was a negative control. Precipitated DNA or 1% of the chromatin input was amplified with primers for the Tnfaip3 promoter (−89 ∼ −410). The PCR products were loaded and separated on a 2% agarose gel. One of two independent experiments is shown. (C) LPS-induced association of p65 and C/EBPβ with Tnfaip3 was reduced in the presence of p38 inhibition. Chromatin isolated from RAW264.7 cells treated with LPS (100 ng/ml) for 4 h in the absence or presence of SB202190 (10 µM) were subjected to ChIP assay as described above. The relative quantity of promoter enriched by ChIP was quantified by real-time PCR and expressed as the fold enrichment of untreated control samples after normalization to rabbit IgG. Data represent the means of two independent experiments. (D) LPS-induced expression of A20 (TNFAIP3) was decreased in C/EBPβ-depleted RAW264.7 cells. Cells were infected with lentiviruses encoding shRNA against luciferase ( shLuc ) or C/EBPβ ( shCebpb ), and treated with LPS (100 ng/ml) for the indicated times. Cell lysates were collected and analyzed by immunoblotting using the indicated antibodies.
Figure Legend Snippet: NF-κB and C/EBPβ are required for induction of Tnfaip3 in LPS-activated macrophages. (A) Schematic map of predicted NF-κB p65 and C/EBPβ DNA binding sites in the promoter of Tnfaip3 . oPOSSUM ( http://www.cisreg.ca/oPOSSUM/ ) was used as the prediction tool. The JASPAR CORE vertebrate database was selected for transcription factor binding site matrices. Two arrows depict the PCR primers. (B) p65 and C/EBPβ bind to the promoter of Tnfaip3 after LPS stimulation. RAW264.7 cells were treated with LPS (100 ng/ml) for the indicated times. Chromatin was immunoprecipitated with anti-p65 and anti-C/EBPβ antibodies. Rabbit IgG was a negative control. Precipitated DNA or 1% of the chromatin input was amplified with primers for the Tnfaip3 promoter (−89 ∼ −410). The PCR products were loaded and separated on a 2% agarose gel. One of two independent experiments is shown. (C) LPS-induced association of p65 and C/EBPβ with Tnfaip3 was reduced in the presence of p38 inhibition. Chromatin isolated from RAW264.7 cells treated with LPS (100 ng/ml) for 4 h in the absence or presence of SB202190 (10 µM) were subjected to ChIP assay as described above. The relative quantity of promoter enriched by ChIP was quantified by real-time PCR and expressed as the fold enrichment of untreated control samples after normalization to rabbit IgG. Data represent the means of two independent experiments. (D) LPS-induced expression of A20 (TNFAIP3) was decreased in C/EBPβ-depleted RAW264.7 cells. Cells were infected with lentiviruses encoding shRNA against luciferase ( shLuc ) or C/EBPβ ( shCebpb ), and treated with LPS (100 ng/ml) for the indicated times. Cell lysates were collected and analyzed by immunoblotting using the indicated antibodies.

Techniques Used: Binding Assay, Polymerase Chain Reaction, Immunoprecipitation, Negative Control, Amplification, Agarose Gel Electrophoresis, Inhibition, Isolation, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Expressing, Infection, shRNA, Luciferase

Depletion of IKKβ expression and inhibition of p38 signaling pathway in Ikkβ Δ and SB202190-treated bone marrow-derived macrophages (BMDMs). (A) Immunoblotting of IKKβ and p38 from BMDMs isolated from wild-type (wt; Ikkβ F/F ) and Ikkβ Δ mice. (B) Immunoblotting of p38 and phosphorylated p38 from wt BMDMs treated with LPS (100 ng/mL) in the absence or presence of SB202190 (10 µM) for 2 h. (C D) mRNA expression levels of IL-1β and IL-6 were inhibited in SB202190-treated BMDMs after LPS treatment. The expression levels of Il1b (C) and Il6 (D) were determined from wt BMDMs treated with LPS (100 ng/mL) for 4 h in the absence or presence of SB202190 (10 µM) for 2 h using real-time RT-PCR. Data represent the mean ± SEM for three independent experiments. ***, P
Figure Legend Snippet: Depletion of IKKβ expression and inhibition of p38 signaling pathway in Ikkβ Δ and SB202190-treated bone marrow-derived macrophages (BMDMs). (A) Immunoblotting of IKKβ and p38 from BMDMs isolated from wild-type (wt; Ikkβ F/F ) and Ikkβ Δ mice. (B) Immunoblotting of p38 and phosphorylated p38 from wt BMDMs treated with LPS (100 ng/mL) in the absence or presence of SB202190 (10 µM) for 2 h. (C D) mRNA expression levels of IL-1β and IL-6 were inhibited in SB202190-treated BMDMs after LPS treatment. The expression levels of Il1b (C) and Il6 (D) were determined from wt BMDMs treated with LPS (100 ng/mL) for 4 h in the absence or presence of SB202190 (10 µM) for 2 h using real-time RT-PCR. Data represent the mean ± SEM for three independent experiments. ***, P

Techniques Used: Expressing, Inhibition, Derivative Assay, Isolation, Mouse Assay, Quantitative RT-PCR

Identification of LPS-induced genes that were regulated by both NF-κB and p38. (A) Venn diagram of NF-κB and p38-dependent genes. NF-κB-related genes were identified from genes that were down-regulated in Ikkβ Δ BMDMs as compared with wt BMDMs after LPS treatment, and p38-related genes were selected by comparing SB202190- (p38-inhibitor) with dimethyl sulfoxide (DMSO)-treated BMDMs after LPS treatment. Thirty-two LPS-induced genes were regulated by both NF-κB and p38-downstream transcription factors. (B) Hierarchical clustering of average fold change for the NF-κB and p38-dependent genes. Each column represents the average fold change 4 h after LPS treatment compared to 0 h. *: genes chosen for PCR validation. (C) Relative fold changes of Il1b, Serpinb2, Tnfaip3 , and Zc3h12a mRNA from BMDMs from wt and Ikkβ Δ cells stimulated with LPS (100 ng/mL) for 4 h in the presence or absence of SB202190 (10 µM) were measured by quantitative RT-PCR. The fold change of 4 h vs. 0 h was normalized to wt BMDMs. The internal control was Cyclophilin A mRNA ( Cypa ). Data represent the mean ± SD for at least two independent experiments. *, P
Figure Legend Snippet: Identification of LPS-induced genes that were regulated by both NF-κB and p38. (A) Venn diagram of NF-κB and p38-dependent genes. NF-κB-related genes were identified from genes that were down-regulated in Ikkβ Δ BMDMs as compared with wt BMDMs after LPS treatment, and p38-related genes were selected by comparing SB202190- (p38-inhibitor) with dimethyl sulfoxide (DMSO)-treated BMDMs after LPS treatment. Thirty-two LPS-induced genes were regulated by both NF-κB and p38-downstream transcription factors. (B) Hierarchical clustering of average fold change for the NF-κB and p38-dependent genes. Each column represents the average fold change 4 h after LPS treatment compared to 0 h. *: genes chosen for PCR validation. (C) Relative fold changes of Il1b, Serpinb2, Tnfaip3 , and Zc3h12a mRNA from BMDMs from wt and Ikkβ Δ cells stimulated with LPS (100 ng/mL) for 4 h in the presence or absence of SB202190 (10 µM) were measured by quantitative RT-PCR. The fold change of 4 h vs. 0 h was normalized to wt BMDMs. The internal control was Cyclophilin A mRNA ( Cypa ). Data represent the mean ± SD for at least two independent experiments. *, P

Techniques Used: Polymerase Chain Reaction, Quantitative RT-PCR

C/EBPβ and A20 (TNFAIP3) were suppressed in Ikkβ Δ and p38-inhibited macrophages. (A–C) Expression levels of Tnfaip3 and Cebpb mRNA were decreased in Ikkβ Δ and p38-inhibited BMDMs in response to LPS. BMDMs from wt and Ikkβ Δ cells treated with or without SB202190 (10 µM) were stimulated with LPS (100 ng/mL) for 4 h. Total RNAs were isolated and analyzed by semi-quantitative RT-PCR (A) or quantitative real-time RT-PCR for expression of Cebpb (B) and Tnfaip3 (C) mRNAs. Results were normalized to Cyclophilin A ( Cypa ) and are presented relative to expression in wt BMDMs. * P
Figure Legend Snippet: C/EBPβ and A20 (TNFAIP3) were suppressed in Ikkβ Δ and p38-inhibited macrophages. (A–C) Expression levels of Tnfaip3 and Cebpb mRNA were decreased in Ikkβ Δ and p38-inhibited BMDMs in response to LPS. BMDMs from wt and Ikkβ Δ cells treated with or without SB202190 (10 µM) were stimulated with LPS (100 ng/mL) for 4 h. Total RNAs were isolated and analyzed by semi-quantitative RT-PCR (A) or quantitative real-time RT-PCR for expression of Cebpb (B) and Tnfaip3 (C) mRNAs. Results were normalized to Cyclophilin A ( Cypa ) and are presented relative to expression in wt BMDMs. * P

Techniques Used: Expressing, Isolation, Quantitative RT-PCR

5) Product Images from "In vitro and in vivo cytotoxicity of troglitazone in pancreatic cancer"

Article Title: In vitro and in vivo cytotoxicity of troglitazone in pancreatic cancer

Journal: Journal of Experimental & Clinical Cancer Research : CR

doi: 10.1186/s13046-017-0557-6

Involvement of Akt and MAPK signaling in TGZ-induced cell death. a Akt and MAPK protein expression. Cells (1.75 × 10 6 ) were pre-cultured for 24 h in 100-mm dishes and treated with TGZ (50 μM) for various durations. Protein (15 μg) was analyzed by western blot for expression of Akt, ERK, JNK, p38, and the phosphorylated forms of each protein. β-Actin was used as a loading control. b Effects of a JNK inhibitor (SP600125) and a p38 inhibitor (SB202190) on TGZ-induced cell death. Cells were pre-cultured for 24 h at density of 1 × 10 4 cells/well in 96-well plates and then exposed to TGZ (50 μM) in the presence or absence of SP600125 or SB202190 (1 and 3 μM, respectively) for 24 h. Cell viability was assessed by fluorescence assay and is expressed as mean + SD ( n = 3–5). Statistical significance was assessed by Dunnett’s test (TGZ vs. TGZ + inhibitors, n.s., not significant). MAPK, mitogen-activated protein kinase; ERK, extracellular signal-regulated kinase; JNK, c-Jun N-terminal kinase; TGZ, troglitazone
Figure Legend Snippet: Involvement of Akt and MAPK signaling in TGZ-induced cell death. a Akt and MAPK protein expression. Cells (1.75 × 10 6 ) were pre-cultured for 24 h in 100-mm dishes and treated with TGZ (50 μM) for various durations. Protein (15 μg) was analyzed by western blot for expression of Akt, ERK, JNK, p38, and the phosphorylated forms of each protein. β-Actin was used as a loading control. b Effects of a JNK inhibitor (SP600125) and a p38 inhibitor (SB202190) on TGZ-induced cell death. Cells were pre-cultured for 24 h at density of 1 × 10 4 cells/well in 96-well plates and then exposed to TGZ (50 μM) in the presence or absence of SP600125 or SB202190 (1 and 3 μM, respectively) for 24 h. Cell viability was assessed by fluorescence assay and is expressed as mean + SD ( n = 3–5). Statistical significance was assessed by Dunnett’s test (TGZ vs. TGZ + inhibitors, n.s., not significant). MAPK, mitogen-activated protein kinase; ERK, extracellular signal-regulated kinase; JNK, c-Jun N-terminal kinase; TGZ, troglitazone

Techniques Used: Expressing, Cell Culture, Western Blot, Fluorescence

6) Product Images from "Oxidized low-density lipoprotein (oxLDL) affects load-free cell shortening of cardiomyocytes in a proprotein convertase subtilisin/kexin 9 (PCSK9)-dependent way"

Article Title: Oxidized low-density lipoprotein (oxLDL) affects load-free cell shortening of cardiomyocytes in a proprotein convertase subtilisin/kexin 9 (PCSK9)-dependent way

Journal: Basic Research in Cardiology

doi: 10.1007/s00395-017-0650-1

Participation of MAPK pathways in oxLDL-dependent effects: a cell shortening of cardiomyocytes exposed to oxLDL (20 µg/ml) and SP600125 (10 µM). b Cell shortening of cardiomyocytes exposed to oxLDL (20 µg/ml) and PD98059 (10 µM). c Cell shortening of cardiomyocytes exposed to oxLDL (20 µg/ml) and SB202190 (10 µM). In a – c a represents p
Figure Legend Snippet: Participation of MAPK pathways in oxLDL-dependent effects: a cell shortening of cardiomyocytes exposed to oxLDL (20 µg/ml) and SP600125 (10 µM). b Cell shortening of cardiomyocytes exposed to oxLDL (20 µg/ml) and PD98059 (10 µM). c Cell shortening of cardiomyocytes exposed to oxLDL (20 µg/ml) and SB202190 (10 µM). In a – c a represents p

Techniques Used:

7) Product Images from "Upregulation of maspin expression in human cervical carcinoma cells by transforming growth factor β1 through the convergence of Smad and non-Smad signaling pathways"

Article Title: Upregulation of maspin expression in human cervical carcinoma cells by transforming growth factor β1 through the convergence of Smad and non-Smad signaling pathways

Journal: Oncology Letters

doi: 10.3892/ol.2017.5939

Induction of maspin expression through Smad and non-Smad signaling pathways. (A) Cultures of HeLa cells were pre-treated with 1, 5 or 10 µM SIS3 prior to the addition of 10 ng/ml TGF-β1 and a 48-h incubation. Maspin, p38 and p-p38 proteins were detected by western blotting. (B) HeLa cells were incubated with 100 µM PDTC, 1 µM wortmannin, 10 µM SP600125, 10 µM U0126 or 10 µM SB202190 for 1 h at 37°C prior to the addition of 10 ng/ml TGF-β1, and then further incubated for 48 h. Maspin, p-Smad2, Smad2, p-p38 and p38 were detected by western blotting. Actin was used for normalization of gel loading. Smad, mothers against decapentaplegic homolog; SIS3, Smad3 inhibitor; TGF-β1, transforming growth factor β1; p-p38, phospho-p38; p-Smad2, phospho-Smad2; PDTC, pyrrolidine dithiocarbamate.
Figure Legend Snippet: Induction of maspin expression through Smad and non-Smad signaling pathways. (A) Cultures of HeLa cells were pre-treated with 1, 5 or 10 µM SIS3 prior to the addition of 10 ng/ml TGF-β1 and a 48-h incubation. Maspin, p38 and p-p38 proteins were detected by western blotting. (B) HeLa cells were incubated with 100 µM PDTC, 1 µM wortmannin, 10 µM SP600125, 10 µM U0126 or 10 µM SB202190 for 1 h at 37°C prior to the addition of 10 ng/ml TGF-β1, and then further incubated for 48 h. Maspin, p-Smad2, Smad2, p-p38 and p38 were detected by western blotting. Actin was used for normalization of gel loading. Smad, mothers against decapentaplegic homolog; SIS3, Smad3 inhibitor; TGF-β1, transforming growth factor β1; p-p38, phospho-p38; p-Smad2, phospho-Smad2; PDTC, pyrrolidine dithiocarbamate.

Techniques Used: Expressing, Incubation, Western Blot

8) Product Images from "TRAIL/DR5 Signaling Promotes Macrophage Foam Cell Formation by Modulating Scavenger Receptor Expression"

Article Title: TRAIL/DR5 Signaling Promotes Macrophage Foam Cell Formation by Modulating Scavenger Receptor Expression

Journal: PLoS ONE

doi: 10.1371/journal.pone.0087059

Role of MAPKs in TRAIL-induced SR-AI expression. ( A ) Western blot showing the effects of TRAIL (10 ng ml −1 ) on phosphorylation of ERK1/2, p38 and JNK MAPKs in RAW264.7 cells. TNF-α (20 ng ml −1 ) was used as a positive control. The total levels of FADD or TRAF2 were not changed. ( B ) Effects of the ERK pathway inhibitor U0126 (1 µM), p38 inhibitor SB202190 (1 µM) and JNK Inhibitor II (1 µM) on TRAIL-stimulated SR-AI expression. The results are expressed as fold of control (Con). * P
Figure Legend Snippet: Role of MAPKs in TRAIL-induced SR-AI expression. ( A ) Western blot showing the effects of TRAIL (10 ng ml −1 ) on phosphorylation of ERK1/2, p38 and JNK MAPKs in RAW264.7 cells. TNF-α (20 ng ml −1 ) was used as a positive control. The total levels of FADD or TRAF2 were not changed. ( B ) Effects of the ERK pathway inhibitor U0126 (1 µM), p38 inhibitor SB202190 (1 µM) and JNK Inhibitor II (1 µM) on TRAIL-stimulated SR-AI expression. The results are expressed as fold of control (Con). * P

Techniques Used: Expressing, Western Blot, Positive Control

9) Product Images from "?5?1 integrin induces the expression of noncartilaginous procollagen gene expression in articular chondrocytes cultured in monolayers"

Article Title: ?5?1 integrin induces the expression of noncartilaginous procollagen gene expression in articular chondrocytes cultured in monolayers

Journal: Arthritis Research & Therapy

doi: 10.1186/ar4307

Phosphoinositide 3-kinase/AKT signaling may be involved in induction of noncartilaginous collagen expression in dedifferentiating chondrocytes. (a) Primary cultured chondrocytes maintained in monolayers for 5 days were treated with specific signal inhibitors for 24 hours, and expression of type I procollagen ( COL1A1 ) and type III procollagen ( COL3A1 ) was evaluated by quantitative PCR. For some cells, expression of type II procollagen ( COL2A1 ) and aggrecan ( ACAN ) was also evaluated. (b) Experiment was repeated using two specific inhibitors for AKT phosphorylation, and expression of the above four genes was evaluated by quantitative PCR. (a) , (b) Inhibitors were used at following concentrations: SB202190, 20 μM; SB203580, 20 μM; PD98059, 20 μM; U0126, 10 μM; SP600125, 10 μM; GF109203X, 5 μM; Wortmannin, 0.5 μM; LY294002, 20 μM; Akt inhibitor IV, 5 μM; Akt inhibitor VIII, 5 μM. Results are shown by relative ratios against control cells treated with dimethylsulfoxide (Control; open bars). Bars represent mean ± standard error of the mean (SEM) of three (b) , five (SB202190, SB203580, U1026 in (a) ) or six (the other inhibitors in (a) ) independent experiments. ** P
Figure Legend Snippet: Phosphoinositide 3-kinase/AKT signaling may be involved in induction of noncartilaginous collagen expression in dedifferentiating chondrocytes. (a) Primary cultured chondrocytes maintained in monolayers for 5 days were treated with specific signal inhibitors for 24 hours, and expression of type I procollagen ( COL1A1 ) and type III procollagen ( COL3A1 ) was evaluated by quantitative PCR. For some cells, expression of type II procollagen ( COL2A1 ) and aggrecan ( ACAN ) was also evaluated. (b) Experiment was repeated using two specific inhibitors for AKT phosphorylation, and expression of the above four genes was evaluated by quantitative PCR. (a) , (b) Inhibitors were used at following concentrations: SB202190, 20 μM; SB203580, 20 μM; PD98059, 20 μM; U0126, 10 μM; SP600125, 10 μM; GF109203X, 5 μM; Wortmannin, 0.5 μM; LY294002, 20 μM; Akt inhibitor IV, 5 μM; Akt inhibitor VIII, 5 μM. Results are shown by relative ratios against control cells treated with dimethylsulfoxide (Control; open bars). Bars represent mean ± standard error of the mean (SEM) of three (b) , five (SB202190, SB203580, U1026 in (a) ) or six (the other inhibitors in (a) ) independent experiments. ** P

Techniques Used: Expressing, Cell Culture, Real-time Polymerase Chain Reaction

10) Product Images from "Cytotoxicity of 15-Deoxy-?12,14-prostaglandin J2 through PPAR?-independent Pathway and the Involvement of the JNK and Akt Pathway in Renal Cell Carcinoma"

Article Title: Cytotoxicity of 15-Deoxy-?12,14-prostaglandin J2 through PPAR?-independent Pathway and the Involvement of the JNK and Akt Pathway in Renal Cell Carcinoma

Journal: International Journal of Medical Sciences

doi: 10.7150/ijms.4455

Involvement of the p38 and JNK MAPK pathway in 15d-PGJ 2 -induced cell death. Effects of a p38 MAPK inhibitor, SB202190 (A), or JNK inhibitor, SP600125 (B, C), on 15d-PGJ 2 -induced cell death and the effects of 15d-PGJ 2 on phosphorylation of JNK (D). Cells were precultured for 24 h at 5 × 10 3 /well in 96-well plates and exposed to 15d-PGJ 2 at approximately the IC 50 in the presence or absence of SB202190 (3 μM) or SP600125 (0.1, 0.3, 1 μM) for 24 h. Cell viability was assessed by fluorescent assay, and data represent the mean ± S.D. from 4 independent preparations. Statistical significance was assessed by t-test or Dunnett's test. To detect proteins, cells were precultured for 24 h in 100-mm dishes. Cells were then treated with 15d-PGJ 2 at 3 μM for 0 and 8 h. Protein (15 μg) was analyzed by Western blotting for the expression of phospho-JNK. Relative protein levels were quantified using ImageJ, and each phospho-JNK signal was normalized to the β-actin signal. The representative bands and the results of densitometric analysis from three independent preparations were described, and data represent the mean ± S.D. from 4 independent preparations.
Figure Legend Snippet: Involvement of the p38 and JNK MAPK pathway in 15d-PGJ 2 -induced cell death. Effects of a p38 MAPK inhibitor, SB202190 (A), or JNK inhibitor, SP600125 (B, C), on 15d-PGJ 2 -induced cell death and the effects of 15d-PGJ 2 on phosphorylation of JNK (D). Cells were precultured for 24 h at 5 × 10 3 /well in 96-well plates and exposed to 15d-PGJ 2 at approximately the IC 50 in the presence or absence of SB202190 (3 μM) or SP600125 (0.1, 0.3, 1 μM) for 24 h. Cell viability was assessed by fluorescent assay, and data represent the mean ± S.D. from 4 independent preparations. Statistical significance was assessed by t-test or Dunnett's test. To detect proteins, cells were precultured for 24 h in 100-mm dishes. Cells were then treated with 15d-PGJ 2 at 3 μM for 0 and 8 h. Protein (15 μg) was analyzed by Western blotting for the expression of phospho-JNK. Relative protein levels were quantified using ImageJ, and each phospho-JNK signal was normalized to the β-actin signal. The representative bands and the results of densitometric analysis from three independent preparations were described, and data represent the mean ± S.D. from 4 independent preparations.

Techniques Used: Fluorescence, Western Blot, Expressing

11) Product Images from "Transcription of Tnfaip3 Is Regulated by NF-?B and p38 via C/EBP? in Activated Macrophages"

Article Title: Transcription of Tnfaip3 Is Regulated by NF-?B and p38 via C/EBP? in Activated Macrophages

Journal: PLoS ONE

doi: 10.1371/journal.pone.0073153

NF-κB and C/EBPβ are required for induction of Tnfaip3 in LPS-activated macrophages. (A) Schematic map of predicted NF-κB p65 and C/EBPβ DNA binding sites in the promoter of Tnfaip3 . oPOSSUM ( http://www.cisreg.ca/oPOSSUM/ ) was used as the prediction tool. The JASPAR CORE vertebrate database was selected for transcription factor binding site matrices. Two arrows depict the PCR primers. (B) p65 and C/EBPβ bind to the promoter of Tnfaip3 after LPS stimulation. RAW264.7 cells were treated with LPS (100 ng/ml) for the indicated times. Chromatin was immunoprecipitated with anti-p65 and anti-C/EBPβ antibodies. Rabbit IgG was a negative control. Precipitated DNA or 1% of the chromatin input was amplified with primers for the Tnfaip3 promoter (−89 ∼ −410). The PCR products were loaded and separated on a 2% agarose gel. One of two independent experiments is shown. (C) LPS-induced association of p65 and C/EBPβ with Tnfaip3 was reduced in the presence of p38 inhibition. Chromatin isolated from RAW264.7 cells treated with LPS (100 ng/ml) for 4 h in the absence or presence of SB202190 (10 µM) were subjected to ChIP assay as described above. The relative quantity of promoter enriched by ChIP was quantified by real-time PCR and expressed as the fold enrichment of untreated control samples after normalization to rabbit IgG. Data represent the means of two independent experiments. (D) LPS-induced expression of A20 (TNFAIP3) was decreased in C/EBPβ-depleted RAW264.7 cells. Cells were infected with lentiviruses encoding shRNA against luciferase ( shLuc ) or C/EBPβ ( shCebpb ), and treated with LPS (100 ng/ml) for the indicated times. Cell lysates were collected and analyzed by immunoblotting using the indicated antibodies.
Figure Legend Snippet: NF-κB and C/EBPβ are required for induction of Tnfaip3 in LPS-activated macrophages. (A) Schematic map of predicted NF-κB p65 and C/EBPβ DNA binding sites in the promoter of Tnfaip3 . oPOSSUM ( http://www.cisreg.ca/oPOSSUM/ ) was used as the prediction tool. The JASPAR CORE vertebrate database was selected for transcription factor binding site matrices. Two arrows depict the PCR primers. (B) p65 and C/EBPβ bind to the promoter of Tnfaip3 after LPS stimulation. RAW264.7 cells were treated with LPS (100 ng/ml) for the indicated times. Chromatin was immunoprecipitated with anti-p65 and anti-C/EBPβ antibodies. Rabbit IgG was a negative control. Precipitated DNA or 1% of the chromatin input was amplified with primers for the Tnfaip3 promoter (−89 ∼ −410). The PCR products were loaded and separated on a 2% agarose gel. One of two independent experiments is shown. (C) LPS-induced association of p65 and C/EBPβ with Tnfaip3 was reduced in the presence of p38 inhibition. Chromatin isolated from RAW264.7 cells treated with LPS (100 ng/ml) for 4 h in the absence or presence of SB202190 (10 µM) were subjected to ChIP assay as described above. The relative quantity of promoter enriched by ChIP was quantified by real-time PCR and expressed as the fold enrichment of untreated control samples after normalization to rabbit IgG. Data represent the means of two independent experiments. (D) LPS-induced expression of A20 (TNFAIP3) was decreased in C/EBPβ-depleted RAW264.7 cells. Cells were infected with lentiviruses encoding shRNA against luciferase ( shLuc ) or C/EBPβ ( shCebpb ), and treated with LPS (100 ng/ml) for the indicated times. Cell lysates were collected and analyzed by immunoblotting using the indicated antibodies.

Techniques Used: Binding Assay, Polymerase Chain Reaction, Immunoprecipitation, Negative Control, Amplification, Agarose Gel Electrophoresis, Inhibition, Isolation, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Expressing, Infection, shRNA, Luciferase

Depletion of IKKβ expression and inhibition of p38 signaling pathway in Ikkβ Δ and SB202190-treated bone marrow-derived macrophages (BMDMs). (A) Immunoblotting of IKKβ and p38 from BMDMs isolated from wild-type (wt; Ikkβ F/F ) and Ikkβ Δ mice. (B) Immunoblotting of p38 and phosphorylated p38 from wt BMDMs treated with LPS (100 ng/mL) in the absence or presence of SB202190 (10 µM) for 2 h. (C D) mRNA expression levels of IL-1β and IL-6 were inhibited in SB202190-treated BMDMs after LPS treatment. The expression levels of Il1b (C) and Il6 (D) were determined from wt BMDMs treated with LPS (100 ng/mL) for 4 h in the absence or presence of SB202190 (10 µM) for 2 h using real-time RT-PCR. Data represent the mean ± SEM for three independent experiments. ***, P
Figure Legend Snippet: Depletion of IKKβ expression and inhibition of p38 signaling pathway in Ikkβ Δ and SB202190-treated bone marrow-derived macrophages (BMDMs). (A) Immunoblotting of IKKβ and p38 from BMDMs isolated from wild-type (wt; Ikkβ F/F ) and Ikkβ Δ mice. (B) Immunoblotting of p38 and phosphorylated p38 from wt BMDMs treated with LPS (100 ng/mL) in the absence or presence of SB202190 (10 µM) for 2 h. (C D) mRNA expression levels of IL-1β and IL-6 were inhibited in SB202190-treated BMDMs after LPS treatment. The expression levels of Il1b (C) and Il6 (D) were determined from wt BMDMs treated with LPS (100 ng/mL) for 4 h in the absence or presence of SB202190 (10 µM) for 2 h using real-time RT-PCR. Data represent the mean ± SEM for three independent experiments. ***, P

Techniques Used: Expressing, Inhibition, Derivative Assay, Isolation, Mouse Assay, Quantitative RT-PCR

Identification of LPS-induced genes that were regulated by both NF-κB and p38. (A) Venn diagram of NF-κB and p38-dependent genes. NF-κB-related genes were identified from genes that were down-regulated in Ikkβ Δ BMDMs as compared with wt BMDMs after LPS treatment, and p38-related genes were selected by comparing SB202190- (p38-inhibitor) with dimethyl sulfoxide (DMSO)-treated BMDMs after LPS treatment. Thirty-two LPS-induced genes were regulated by both NF-κB and p38-downstream transcription factors. (B) Hierarchical clustering of average fold change for the NF-κB and p38-dependent genes. Each column represents the average fold change 4 h after LPS treatment compared to 0 h. *: genes chosen for PCR validation. (C) Relative fold changes of Il1b, Serpinb2, Tnfaip3 , and Zc3h12a mRNA from BMDMs from wt and Ikkβ Δ cells stimulated with LPS (100 ng/mL) for 4 h in the presence or absence of SB202190 (10 µM) were measured by quantitative RT-PCR. The fold change of 4 h vs. 0 h was normalized to wt BMDMs. The internal control was Cyclophilin A mRNA ( Cypa ). Data represent the mean ± SD for at least two independent experiments. *, P
Figure Legend Snippet: Identification of LPS-induced genes that were regulated by both NF-κB and p38. (A) Venn diagram of NF-κB and p38-dependent genes. NF-κB-related genes were identified from genes that were down-regulated in Ikkβ Δ BMDMs as compared with wt BMDMs after LPS treatment, and p38-related genes were selected by comparing SB202190- (p38-inhibitor) with dimethyl sulfoxide (DMSO)-treated BMDMs after LPS treatment. Thirty-two LPS-induced genes were regulated by both NF-κB and p38-downstream transcription factors. (B) Hierarchical clustering of average fold change for the NF-κB and p38-dependent genes. Each column represents the average fold change 4 h after LPS treatment compared to 0 h. *: genes chosen for PCR validation. (C) Relative fold changes of Il1b, Serpinb2, Tnfaip3 , and Zc3h12a mRNA from BMDMs from wt and Ikkβ Δ cells stimulated with LPS (100 ng/mL) for 4 h in the presence or absence of SB202190 (10 µM) were measured by quantitative RT-PCR. The fold change of 4 h vs. 0 h was normalized to wt BMDMs. The internal control was Cyclophilin A mRNA ( Cypa ). Data represent the mean ± SD for at least two independent experiments. *, P

Techniques Used: Polymerase Chain Reaction, Quantitative RT-PCR

C/EBPβ and A20 (TNFAIP3) were suppressed in Ikkβ Δ and p38-inhibited macrophages. (A–C) Expression levels of Tnfaip3 and Cebpb mRNA were decreased in Ikkβ Δ and p38-inhibited BMDMs in response to LPS. BMDMs from wt and Ikkβ Δ cells treated with or without SB202190 (10 µM) were stimulated with LPS (100 ng/mL) for 4 h. Total RNAs were isolated and analyzed by semi-quantitative RT-PCR (A) or quantitative real-time RT-PCR for expression of Cebpb (B) and Tnfaip3 (C) mRNAs. Results were normalized to Cyclophilin A ( Cypa ) and are presented relative to expression in wt BMDMs. * P
Figure Legend Snippet: C/EBPβ and A20 (TNFAIP3) were suppressed in Ikkβ Δ and p38-inhibited macrophages. (A–C) Expression levels of Tnfaip3 and Cebpb mRNA were decreased in Ikkβ Δ and p38-inhibited BMDMs in response to LPS. BMDMs from wt and Ikkβ Δ cells treated with or without SB202190 (10 µM) were stimulated with LPS (100 ng/mL) for 4 h. Total RNAs were isolated and analyzed by semi-quantitative RT-PCR (A) or quantitative real-time RT-PCR for expression of Cebpb (B) and Tnfaip3 (C) mRNAs. Results were normalized to Cyclophilin A ( Cypa ) and are presented relative to expression in wt BMDMs. * P

Techniques Used: Expressing, Isolation, Quantitative RT-PCR

12) Product Images from "Epinephrine modulates Na+/K+ ATPase activity in Caco-2 cells via Src, p38MAPK, ERK and PGE2"

Article Title: Epinephrine modulates Na+/K+ ATPase activity in Caco-2 cells via Src, p38MAPK, ERK and PGE2

Journal: PLoS ONE

doi: 10.1371/journal.pone.0193139

(A) Effect of epinephrine (0.5 mM, 20min) and (B) PGE2 (1nM,20min) on the activity of the Na + /K + ATPase when p38MAPK was inhibited with SB202190 (50μM, added 15 min before epinephrine or PGE2). Values are means ± SEM of 3 observations. Bars not sharing the same letter are considered significantly different from each other at p
Figure Legend Snippet: (A) Effect of epinephrine (0.5 mM, 20min) and (B) PGE2 (1nM,20min) on the activity of the Na + /K + ATPase when p38MAPK was inhibited with SB202190 (50μM, added 15 min before epinephrine or PGE2). Values are means ± SEM of 3 observations. Bars not sharing the same letter are considered significantly different from each other at p

Techniques Used: Activity Assay

13) Product Images from "p38?, A Novel Regulatory Target of Pokemon in Hepatic Cells"

Article Title: p38?, A Novel Regulatory Target of Pokemon in Hepatic Cells

Journal: International Journal of Molecular Sciences

doi: 10.3390/ijms140713511

( a ) Inhibition of SB202190 on cell viability: MTT assays in HepG2, BEL7404 and HL7702 cells treated with SB202190 for 48 h at different concentrations (0, 2.5, 5, 10, 25 and 50 μM); ( b ) Western blot: Displaying that SB202190 dose-dependently inhibits the phosphorylation of p38 downstream proteins. HepG2 cells were treated with SB202190 for 24 h at different concentrations (0, 10, 25 and 50 μM); ( c – g ) HepG2 cells were treated with 25 μM SB202190 at 24 h after transfecting with pcDNA3.1(−)-Pokemon or pcDNA3.1(−): ( c ) HepG2 Cell growth rate; ( d ) Effect of Pokemon and p38 inhibitor SB202190 on colony formation in HepG2 cells, the colony formation rate stands for the proportion of final clone number accounted for in plated cell number; ( e ) In vitro migration assays; ( f ) In vitro invasion assays. Bar chart below the photo stands for the relative fold of the migrated or invaded cell number compared to the negative control group; ( g ) Pokemon activates p38 signaling pathway in hepatic cells: Left panel is Western blot bands. Western blot in HepG2 cells after Pokemon was overexpressed for 60 h, and the cells were treated by SB202190 at the concentration of 25 μM; right panel is quantification of western blot data. * p
Figure Legend Snippet: ( a ) Inhibition of SB202190 on cell viability: MTT assays in HepG2, BEL7404 and HL7702 cells treated with SB202190 for 48 h at different concentrations (0, 2.5, 5, 10, 25 and 50 μM); ( b ) Western blot: Displaying that SB202190 dose-dependently inhibits the phosphorylation of p38 downstream proteins. HepG2 cells were treated with SB202190 for 24 h at different concentrations (0, 10, 25 and 50 μM); ( c – g ) HepG2 cells were treated with 25 μM SB202190 at 24 h after transfecting with pcDNA3.1(−)-Pokemon or pcDNA3.1(−): ( c ) HepG2 Cell growth rate; ( d ) Effect of Pokemon and p38 inhibitor SB202190 on colony formation in HepG2 cells, the colony formation rate stands for the proportion of final clone number accounted for in plated cell number; ( e ) In vitro migration assays; ( f ) In vitro invasion assays. Bar chart below the photo stands for the relative fold of the migrated or invaded cell number compared to the negative control group; ( g ) Pokemon activates p38 signaling pathway in hepatic cells: Left panel is Western blot bands. Western blot in HepG2 cells after Pokemon was overexpressed for 60 h, and the cells were treated by SB202190 at the concentration of 25 μM; right panel is quantification of western blot data. * p

Techniques Used: Inhibition, MTT Assay, Western Blot, In Vitro, Migration, Negative Control, Concentration Assay

14) Product Images from "Epinephrine modulates Na+/K+ ATPase activity in Caco-2 cells via Src, p38MAPK, ERK and PGE2"

Article Title: Epinephrine modulates Na+/K+ ATPase activity in Caco-2 cells via Src, p38MAPK, ERK and PGE2

Journal: PLoS ONE

doi: 10.1371/journal.pone.0193139

(A) Effect of epinephrine (0.5 mM, 20min) and (B) PGE2 (1nM,20min) on the activity of the Na + /K + ATPase when p38MAPK was inhibited with SB202190 (50μM, added 15 min before epinephrine or PGE2). Values are means ± SEM of 3 observations. Bars not sharing the same letter are considered significantly different from each other at p
Figure Legend Snippet: (A) Effect of epinephrine (0.5 mM, 20min) and (B) PGE2 (1nM,20min) on the activity of the Na + /K + ATPase when p38MAPK was inhibited with SB202190 (50μM, added 15 min before epinephrine or PGE2). Values are means ± SEM of 3 observations. Bars not sharing the same letter are considered significantly different from each other at p

Techniques Used: Activity Assay

Related Articles

other:

Article Title: p38?, A Novel Regulatory Target of Pokemon in Hepatic Cells
Article Snippet: Through the attenuation of the promotion by SB202190, we uncovered a new mechanism that Pokemon is involved in.

Article Title: CAY10593 inhibits the human P2X7 receptor independently of phospholipase D1 stimulation
Article Snippet: Rottlerin, LY294002, SB202190, SB203580, U0126 and SP600125 were from Merck Chemicals (Darmstadt, Germany).

Article Title: Involvement of MAPK signalling in radioadaptive response in BALB/c mice exposed to low dose ionizing radiation.
Article Snippet: To investigate low dose ionizing radiation (LDIR)-induced adaptive response in lymphocytes of BALB/c mice and to elucidate related molecular mechanisms.

Article Title: Transcription of Tnfaip3 Is Regulated by NF-?B and p38 via C/EBP? in Activated Macrophages
Article Snippet: The results revealed that both Il1b ( ) and Il6 ( ) mRNAs were significantly (P < 0.005) suppressed in BMDMs pretreated with SB202190.

MTT Assay:

Article Title: p38?, A Novel Regulatory Target of Pokemon in Hepatic Cells
Article Snippet: Antibodies used are as follows: Anti-Pokemon antibody (Abcam, Cambridge, UK) and Goat Control IgG antibody (Abcam). .. MTT Assay Cells were seeded at a density of 3 × 103 /100 μL medium in 96-well plate and treated with the SB202190 (Merck Millipore, Darmstadt, Germany) at different time points, ranging from 24 to 120 h. Cells were incubated with MTT (5 mg/mL) for 4 h and formazan precipitate was dissolved in 100 μL DMSO and the absorbance at 595 nm was measured by Multimode Detector DTX880 (Beckman Coulter, Atlanta, GA, USA). .. Colony Formation Assay HepG2 cells at the exponential phase were plated into 24-well plate (200–300 cells/well) and allowed to adhere for 12 h before treatment.

Incubation:

Article Title: p38?, A Novel Regulatory Target of Pokemon in Hepatic Cells
Article Snippet: Antibodies used are as follows: Anti-Pokemon antibody (Abcam, Cambridge, UK) and Goat Control IgG antibody (Abcam). .. MTT Assay Cells were seeded at a density of 3 × 103 /100 μL medium in 96-well plate and treated with the SB202190 (Merck Millipore, Darmstadt, Germany) at different time points, ranging from 24 to 120 h. Cells were incubated with MTT (5 mg/mL) for 4 h and formazan precipitate was dissolved in 100 μL DMSO and the absorbance at 595 nm was measured by Multimode Detector DTX880 (Beckman Coulter, Atlanta, GA, USA). .. Colony Formation Assay HepG2 cells at the exponential phase were plated into 24-well plate (200–300 cells/well) and allowed to adhere for 12 h before treatment.

Inhibition:

Article Title: Transcription of Tnfaip3 Is Regulated by NF-?B and p38 via C/EBP? in Activated Macrophages
Article Snippet: BMDMs were then collected and cultured in DMEM with 10 ng/ml macrophage colony-stimulating factor for further experiments. .. For inhibition of p38, BMDMs from C57BL/6 mice were treated with 10 µM SB202190 (Merck, Germany) for 2 h prior to use. .. In addition, the murine macrophage-like RAW264.7 cells (ATCC #TIB-71) were maintained in complete DMEM at 37°C in a 5% CO2 humidified incubator.

Mouse Assay:

Article Title: Transcription of Tnfaip3 Is Regulated by NF-?B and p38 via C/EBP? in Activated Macrophages
Article Snippet: BMDMs were then collected and cultured in DMEM with 10 ng/ml macrophage colony-stimulating factor for further experiments. .. For inhibition of p38, BMDMs from C57BL/6 mice were treated with 10 µM SB202190 (Merck, Germany) for 2 h prior to use. .. In addition, the murine macrophage-like RAW264.7 cells (ATCC #TIB-71) were maintained in complete DMEM at 37°C in a 5% CO2 humidified incubator.

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    Merck KGaA sb202190
    Participation of MAPK pathways in oxLDL-dependent effects: a cell shortening of cardiomyocytes exposed to oxLDL (20 µg/ml) and SP600125 (10 µM). b Cell shortening of cardiomyocytes exposed to oxLDL (20 µg/ml) and PD98059 (10 µM). c Cell shortening of cardiomyocytes exposed to oxLDL (20 µg/ml) and <t>SB202190</t> (10 µM). In a – c a represents p
    Sb202190, supplied by Merck KGaA, 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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    Merck KGaA p38 mapk inhibitor sb202190
    Targeting of Dsg3 leads to <t>p38</t> <t>MAPK-dependent</t> loss of cell cohesion. A , targeting of Dsg3 function by either incubation with AK23 or siRNA-mediated depletion of Dsg3 protein levels in HaCaT cells resulted in increased cell monolayer fragmentation in dispase-based
    P38 Mapk Inhibitor Sb202190, supplied by Merck KGaA, 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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    Merck KGaA p38 inhibitor sb202190
    <t>p38</t> inhibitor treatment reverses RNASE1 promoter deacetylation and CHD4 recruitment in inflamed human ECs. HUVEC were pretreated with 20 μM p38 inhibitor (inh.) SB202190 (+) or DMSO as solvent control (–) for 2 h, followed by 10 min TNF-α stimulation [10 ng/ml] (white bars) or left untreated as control (CTRL; black bars). Immunoprecipitation using specific antibodies against histone 4 acetylation (H4ac; left panels), histone 3 lysine 27 acetylation (H3K27ac; middle panels), chromodomain helicase DNA binding protein 4 (CHD4; right panels) or an unspecific IgG control was performed. (A) Region A , (B) Region B , (C) Region C of the RNASE1 promoter were pulled down by the respective antibodies and analyzed by qPCR using respective primers. Results were depicted as % input and the respective control sample with the specific antibody was set to 1. n = 3–4; mean ± SD; Two-way ANOVA was performed using Holm-Sidak post-test. *IgG vs. specific (spec.) antibody (AB): * p
    P38 Inhibitor Sb202190, supplied by Merck KGaA, 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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    Participation of MAPK pathways in oxLDL-dependent effects: a cell shortening of cardiomyocytes exposed to oxLDL (20 µg/ml) and SP600125 (10 µM). b Cell shortening of cardiomyocytes exposed to oxLDL (20 µg/ml) and PD98059 (10 µM). c Cell shortening of cardiomyocytes exposed to oxLDL (20 µg/ml) and SB202190 (10 µM). In a – c a represents p

    Journal: Basic Research in Cardiology

    Article Title: Oxidized low-density lipoprotein (oxLDL) affects load-free cell shortening of cardiomyocytes in a proprotein convertase subtilisin/kexin 9 (PCSK9)-dependent way

    doi: 10.1007/s00395-017-0650-1

    Figure Lengend Snippet: Participation of MAPK pathways in oxLDL-dependent effects: a cell shortening of cardiomyocytes exposed to oxLDL (20 µg/ml) and SP600125 (10 µM). b Cell shortening of cardiomyocytes exposed to oxLDL (20 µg/ml) and PD98059 (10 µM). c Cell shortening of cardiomyocytes exposed to oxLDL (20 µg/ml) and SB202190 (10 µM). In a – c a represents p

    Article Snippet: Actinomycin D, cycloheximide, PD98059, SB202190, and SP600125 were purchased from Merck KGaA (Darmstadt, Germany).

    Techniques:

    Targeting of Dsg3 leads to p38 MAPK-dependent loss of cell cohesion. A , targeting of Dsg3 function by either incubation with AK23 or siRNA-mediated depletion of Dsg3 protein levels in HaCaT cells resulted in increased cell monolayer fragmentation in dispase-based

    Journal: The Journal of Biological Chemistry

    Article Title: Desmoglein 2 Compensates for Desmoglein 3 but Does Not Control Cell Adhesion via Regulation of p38 Mitogen-activated Protein Kinase in Keratinocytes

    doi: 10.1074/jbc.M113.489336

    Figure Lengend Snippet: Targeting of Dsg3 leads to p38 MAPK-dependent loss of cell cohesion. A , targeting of Dsg3 function by either incubation with AK23 or siRNA-mediated depletion of Dsg3 protein levels in HaCaT cells resulted in increased cell monolayer fragmentation in dispase-based

    Article Snippet: The specific p38 MAPK inhibitor SB202190 (Merck, Darmstadt, Germany) was applied at a concentration of 30 μmol/liter for 24 h either alone or 1 h before AK23 incubation started.

    Techniques: Incubation

    p38 MAPK is activated following Dsg3 depletion and forms a complex with Dsg3. A , HaCaT cells were transfected with siRNA targeting either Dsg2 or Dsg3, and successful knockdown was proven by reduced protein levels in a Western blot analysis against non-targeting

    Journal: The Journal of Biological Chemistry

    Article Title: Desmoglein 2 Compensates for Desmoglein 3 but Does Not Control Cell Adhesion via Regulation of p38 Mitogen-activated Protein Kinase in Keratinocytes

    doi: 10.1074/jbc.M113.489336

    Figure Lengend Snippet: p38 MAPK is activated following Dsg3 depletion and forms a complex with Dsg3. A , HaCaT cells were transfected with siRNA targeting either Dsg2 or Dsg3, and successful knockdown was proven by reduced protein levels in a Western blot analysis against non-targeting

    Article Snippet: The specific p38 MAPK inhibitor SB202190 (Merck, Darmstadt, Germany) was applied at a concentration of 30 μmol/liter for 24 h either alone or 1 h before AK23 incubation started.

    Techniques: Transfection, Western Blot

    Dsg3 forms a complex with p-p38 MAPK in both the Triton X-100-soluble and Triton X-100-insoluble pool. Western blot analysis following Triton X-100-mediated cell fractionation in HaCaT cells revealed that Dsg3 was present in the soluble ( S ) and insoluble

    Journal: The Journal of Biological Chemistry

    Article Title: Desmoglein 2 Compensates for Desmoglein 3 but Does Not Control Cell Adhesion via Regulation of p38 Mitogen-activated Protein Kinase in Keratinocytes

    doi: 10.1074/jbc.M113.489336

    Figure Lengend Snippet: Dsg3 forms a complex with p-p38 MAPK in both the Triton X-100-soluble and Triton X-100-insoluble pool. Western blot analysis following Triton X-100-mediated cell fractionation in HaCaT cells revealed that Dsg3 was present in the soluble ( S ) and insoluble

    Article Snippet: The specific p38 MAPK inhibitor SB202190 (Merck, Darmstadt, Germany) was applied at a concentration of 30 μmol/liter for 24 h either alone or 1 h before AK23 incubation started.

    Techniques: Western Blot, Cell Fractionation

    Mechanisms of Dsg2- and Dsg3-mediated cell-cell adhesion in keratinocytes. Targeting of Dsg3 binding either by pemphigus antibodies or siRNA leads to p38 MAPK activation and following retraction of the keratin filament network, which both negatively affect

    Journal: The Journal of Biological Chemistry

    Article Title: Desmoglein 2 Compensates for Desmoglein 3 but Does Not Control Cell Adhesion via Regulation of p38 Mitogen-activated Protein Kinase in Keratinocytes

    doi: 10.1074/jbc.M113.489336

    Figure Lengend Snippet: Mechanisms of Dsg2- and Dsg3-mediated cell-cell adhesion in keratinocytes. Targeting of Dsg3 binding either by pemphigus antibodies or siRNA leads to p38 MAPK activation and following retraction of the keratin filament network, which both negatively affect

    Article Snippet: The specific p38 MAPK inhibitor SB202190 (Merck, Darmstadt, Germany) was applied at a concentration of 30 μmol/liter for 24 h either alone or 1 h before AK23 incubation started.

    Techniques: Binding Assay, Activation Assay

    Dsg3 depletion recruits Dsg2 and p-p38 MAPK to the plasma membrane, accompanied by alterations in PG protein and Dsg2 mRNA expression. A , representative Western blot analysis with detection of Dsg2 and PG in total cell lysates of MEK cells 24 h after

    Journal: The Journal of Biological Chemistry

    Article Title: Desmoglein 2 Compensates for Desmoglein 3 but Does Not Control Cell Adhesion via Regulation of p38 Mitogen-activated Protein Kinase in Keratinocytes

    doi: 10.1074/jbc.M113.489336

    Figure Lengend Snippet: Dsg3 depletion recruits Dsg2 and p-p38 MAPK to the plasma membrane, accompanied by alterations in PG protein and Dsg2 mRNA expression. A , representative Western blot analysis with detection of Dsg2 and PG in total cell lysates of MEK cells 24 h after

    Article Snippet: The specific p38 MAPK inhibitor SB202190 (Merck, Darmstadt, Germany) was applied at a concentration of 30 μmol/liter for 24 h either alone or 1 h before AK23 incubation started.

    Techniques: Expressing, Western Blot

    p38 inhibitor treatment reverses RNASE1 promoter deacetylation and CHD4 recruitment in inflamed human ECs. HUVEC were pretreated with 20 μM p38 inhibitor (inh.) SB202190 (+) or DMSO as solvent control (–) for 2 h, followed by 10 min TNF-α stimulation [10 ng/ml] (white bars) or left untreated as control (CTRL; black bars). Immunoprecipitation using specific antibodies against histone 4 acetylation (H4ac; left panels), histone 3 lysine 27 acetylation (H3K27ac; middle panels), chromodomain helicase DNA binding protein 4 (CHD4; right panels) or an unspecific IgG control was performed. (A) Region A , (B) Region B , (C) Region C of the RNASE1 promoter were pulled down by the respective antibodies and analyzed by qPCR using respective primers. Results were depicted as % input and the respective control sample with the specific antibody was set to 1. n = 3–4; mean ± SD; Two-way ANOVA was performed using Holm-Sidak post-test. *IgG vs. specific (spec.) antibody (AB): * p

    Journal: Frontiers in Cell and Developmental Biology

    Article Title: p38 and Casein Kinase 2 Mediate Ribonuclease 1 Repression in Inflamed Human Endothelial Cells via Promoter Remodeling Through Nucleosome Remodeling and Deacetylase Complex

    doi: 10.3389/fcell.2020.563604

    Figure Lengend Snippet: p38 inhibitor treatment reverses RNASE1 promoter deacetylation and CHD4 recruitment in inflamed human ECs. HUVEC were pretreated with 20 μM p38 inhibitor (inh.) SB202190 (+) or DMSO as solvent control (–) for 2 h, followed by 10 min TNF-α stimulation [10 ng/ml] (white bars) or left untreated as control (CTRL; black bars). Immunoprecipitation using specific antibodies against histone 4 acetylation (H4ac; left panels), histone 3 lysine 27 acetylation (H3K27ac; middle panels), chromodomain helicase DNA binding protein 4 (CHD4; right panels) or an unspecific IgG control was performed. (A) Region A , (B) Region B , (C) Region C of the RNASE1 promoter were pulled down by the respective antibodies and analyzed by qPCR using respective primers. Results were depicted as % input and the respective control sample with the specific antibody was set to 1. n = 3–4; mean ± SD; Two-way ANOVA was performed using Holm-Sidak post-test. *IgG vs. specific (spec.) antibody (AB): * p

    Article Snippet: For inhibitor assays, HUVEC were pretreated for 1 h with the NF-κB inhibitor BAY11-7082 [1 μM, 5 μM], the JNK inhibitor JNK inhibitor II [10 μM, 30 μM], the p38 inhibitor SB202190 [10 μM, 20 μM] (Merck KGaA, Sigma Aldrich, Darmstadt, HE, Germany) prior to indicated stimulation for 24 h. Dimethyl sulfoxide (DMSO) (Carl Roth GmbH & Co., KG, Karlsruhe, BW, Germany) was used as solvent control.

    Techniques: Immunoprecipitation, Binding Assay, Real-time Polymerase Chain Reaction