sb202190  (Enzo Biochem)

Journal: Arthritis Research & Therapy

Article Title: Cytokine signaling-1 suppressor is inducible by IL-1beta and inhibits the catabolic effects of IL-1beta in chondrocytes: its implication in the paradoxical joint-protective role of IL-1beta

doi: 10.1186/ar4381

Figure Lengend Snippet: Effects of SOCS1 on MAP kinase signaling. SW1353 cells were transfected with SOCS1 or shSOCS1 vectors to overexpress or inhibit SOCS1, as described in Methods. A representative immunoblot image showed that SOCS1 overexpression decreased phosphorylation of p38 and JNK, whereas SOCS1 knockdown increased their phosphorylation in the presence of IL-1β (10 ng/ml, A) . The relative proportions of phosphorylated to total protein were determined with densitometry by using Image J software (version 1.48c, [ 22 ]; B) . Data were expressed as the means ± SEM ( n = 3). OE, overexpression; KD, knockdown.

Article Snippet: A p38 MAP kinase inhibitor SB202190 and NF-κB inhibitor SN50 were purchased from Alexis Biochemicals (Farmingdale, MI, USA).

Techniques: Transfection, Over Expression, Software

Journal: Arthritis Research & Therapy

Article Title: Cytokine signaling-1 suppressor is inducible by IL-1beta and inhibits the catabolic effects of IL-1beta in chondrocytes: its implication in the paradoxical joint-protective role of IL-1beta

doi: 10.1186/ar4381

Figure Lengend Snippet: Effect of MAP kinase and NF-κB inhibitors on MMPs secretion from SOCS1-knockdown SW1353 cells. SOCS1-knockdown SW1353 cells were pretreated with inhibitors for 1 hour before stimulation with 10 ng/ml of IL-1β. After 24 hours, the levels of MMP-1, -3, and -13 were dramatically decreased by SB202190, a p38 MAP kinase inhibitor (A) . Blockade of C-JNK (B) and ERK (C) dose-dependently suppressed the secretion of MMPs from shSOCS1-transfected SW1353 cells. The production of MMP-1 and MMP-13 was partially inhibited with the SN50, a specific NF-κB inhibitory peptide (D) . Data were expressed as the mean ± SEM of relative MMPs levels, as compared with the control without inhibitors ( n = 3). *P

Article Snippet: A p38 MAP kinase inhibitor SB202190 and NF-κB inhibitor SN50 were purchased from Alexis Biochemicals (Farmingdale, MI, USA).

Techniques: Transfection

Journal: Scientific Reports

Article Title: Phosphorylation of steroid receptor coactivator-3 (SRC-3) at serine 857 is regulated by the p38MAPK-MK2 axis and affects NF-κB-mediated transcription

doi: 10.1038/s41598-020-68219-4

Figure Lengend Snippet: Activation of p38 MAPK and MK2 is required for efficient nuclear translocation of SRC-3 in response to TNF-α. ( A , B ) p38 MAPK and MK2 is involved in nuclear translocation of SRC-3. A549 WT cells were seeded on coverslip and left overnight. The next day, cells were treated with either DMSO, 10 ng/ml TNF-α or SB-202190 ( A ) or 10 μM PF-3644022 ( B ) separately, or in combination with SB-202190 ( A ) or PF-3644022 ( B ) for 30 min followed by TNF-α stimulation for 60 min. Representative images of the SRC-3 WT A549 cells stained for SRC-3 (red) using anti-SRC-3 antibody and nucleus ( blue, DAPI). The specificity of the antibody for SRC-3 was verified using SRC-3 KO cells (Supplementary Fig. S2 A,B). ( C ) Generation of SRC-3 KO A549 cells. Expression of SRC-3 and actin in SRC-3 WT and SRC-3 KO A549 cells were analyzed by Western-blotting. ( D ) SRC-3 WT is more efficiently translocated into nucleus than SRC-3 S857A in response to TNF-α. SRC-3 KO A549 cells were seeded in 24 well plate and left overnight. The next day, the cells were transfected with 200 ng of vector expressing either SRC-3 wild type (WT)-FLAG or SRC-3 S857A-FLAG. After 48 h, the cells were either stimulated with 10 ng/ml TNF-α for 60 min or left unstimulated. Representative images of the SRC-3 KO A549 cells stained for SRC-3 (red, anti-SRC-3) and nucleus (blue, DAPI). ( E – G ) Quantitative presentation of the distribution of SRC-3 in conditions described in ( A , B , D ) respectively. The cellular localization of SRC-3 was determined as either cytoplasmic and nuclear or mainly nuclear. The SRC-3 overlapping nucleus (DAPI) is considered nuclear and the SRC-3 overlapping the nucleus and present around and outside the nucleus is considered cytoplasmic + nuclear. For quantification, minimum 100 cells were counted for each condition described in ( A , B , D ) and expressed in percentage. Data in ( E , F , G ) are presented as mean ± SD of three replicates. Unpaired t-test was used for analysis of significance between groups compared in the figure. * P

Article Snippet: SB-202190 (#BML-EI294-001), PD-184352 (#ALX-270-471) were purchased from Alexis Biochemicals, CA, USA.

Techniques: Activation Assay, Translocation Assay, Staining, Expressing, Western Blot, Transfection, Plasmid Preparation

Journal: Scientific Reports

Article Title: Phosphorylation of steroid receptor coactivator-3 (SRC-3) at serine 857 is regulated by the p38MAPK-MK2 axis and affects NF-κB-mediated transcription

doi: 10.1038/s41598-020-68219-4

Figure Lengend Snippet: SRC-3 is required for MK2-mediated induction of IL-6 expression in response to TNF-α. ( A , B ) SRC-3 is involved in NF-κB activation. ( A ) SRC-3 KO A549 cells were co-transfected with 120 ng κB-ConA-luc vector and 50 ng of either pSG5 empty vector, SRC-3 wild type (WT)-FLAG or SRC-3 S857A-FLAG vector. After 48 h TNF-α was added (if not other indicated 10 ng/ml for 5 h) before determination of luciferase activity relative to pSG5. ( B ) A549-NF-κB-Luc cells were transfected with scrambled siRNA or SRC-3 siRNA and 48 h later stimulated with TNF-α or left unstimulated. Nontransfected cells were pretreated with PF-3644022 for 30 min before TNF-α treatment. Luciferase activities are shown relative to unstimulated scrambled siRNA. ( C , D ) SRC-3 is involved in TNF-α-induced IL-6 expression. SRC-3 WT and SRC-3 KO A549 cells were stimulated with TNF-α for 2 h or left unstimulated. mRNA expression of IL-6 ( C ) and MMP9 ( D ) were determined relative to GAPDH and TFRC. Fold changes are presented relative to unstimulated SRC-3 WT cells. ( E , J ) MK2 and p38 MAPK activity are required for transcription of IL-6. A549 cells were transfected with 120 ng pGL3-IL-6-promoter vector and after 48 h treated for 30 min with 0.2 μl DMSO, 10 μM PF-3644022 ( E ) or SB-202190 ( J ) followed by stimulation with TNF-α. Luciferase activities are shown relative to DMSO. ( F – H ) MK2 is involved in TNF-α induced TRAF1 ( F ), IL-8 ( G ) and ICAM1 ( H ) mRNA expression. A549 cells pretreated with DMSO or 10 μM PF-3644022 for 30 min were stimulated with TNF-α for 2 h or left unstimulated. MRNA expression were determined relative to GAPDH and TFRC. Fold changes are presented relative to DMSO. ( I ) p38 MAPK is involved in NF-κB-dependent luciferase activity. A549-NF-κB-Luc cells were pretreated with DMSO or SB-202190 and then stimulated with TNF-α or left unstimulated. Luciferase activities are shown relative to DMSO. ( K – M ) p38 MAPK is involved in TNF-α-induced IL-6 ( K ) and IL-8 ( L ) but not MMP9 ( M ) mRNA expression. A549 cells pretreated with DMSO or 10 μM SB-202190 for 30 min were stimulated with TNF-α or left unstimulated. MRNA expression were determined relative to GAPDH and TFRC. Fold changes are presented relative to DMSO. Data are presented as mean ± SD (n = 3). Unpaired t-test; * P

Article Snippet: SB-202190 (#BML-EI294-001), PD-184352 (#ALX-270-471) were purchased from Alexis Biochemicals, CA, USA.

Techniques: Expressing, Activation Assay, Transfection, Plasmid Preparation, Luciferase, Activity Assay

Journal: Scientific Reports

Article Title: Phosphorylation of steroid receptor coactivator-3 (SRC-3) at serine 857 is regulated by the p38MAPK-MK2 axis and affects NF-κB-mediated transcription

doi: 10.1038/s41598-020-68219-4

Figure Lengend Snippet: Activation of p38 MAPK results in phosphorylation of SRC-3 at S857. ( A ) p38 MAPK but not ERK1/2 is involved in phosphorylation of SRC-3 at S857. H1299 cells were incubated with either 10 μM MEK1/2 inhibitor (PD-184352) or 10 μM p38 MAPK inhibitor (SB-202190) for 2 h before SRC-3 was immunoprecipitated (IP). The IP lysate and whole cell extract (WCE) were analyzed by Western-blotting using anti-P-S857-SRC-3, anti-SRC-3, anti-phospho ERK1/2 MAPK and anti-ERK2 antibodies. ( B – F ) p38 MAPK activation phosphorylates SRC-3 at S857. The full-length blots are presented in supplementary figure S12 . H1299 ( B ), A549 ( C ), HEK 293 ( D ), HeLa ( E ) and MDA MB 231 ( F ) cells were stimulated with either 10 ng/ml TNF-α (15 min), 10 μg/ml anisomycin or 250 μM sodium arsenite (SA) for 30 min. Unstimulated cells were used as control. The cells were lysed and the level of phosphorylation of SRC-3 at S857 and p38 MAPK at T180/Y182 was analyzed by Western-blotting using anti-P-S857-SRC-3, anti-SRC-3, anti-phospho-p38 MAPK and anti-p38 MAPK antibodies. The full-length blots are presented in supplementary figures S13 – S17 . ( G , H ) Inhibition of p38 MAPK activation prevents TNF-α and anisomycin-induced phosphorylation of SRC-3 at S857. A549 cells were seeded and left overnight. On the other day, the cells were pretreated either with DMSO or 10 μM SB-202190 for 30 min. Then they were stimulated with 10 ng/ml TNF-α (15 min) or 10 μg/ml anisomycin ( G ) or 500 μM sodium arsenite (SA) ( H ) for 30 min. Finally, the cells were lysed and level of phosphorylation of SRC-3 at S857, HSP27 at S82, total amount of SRC-3, HSP27 and actin were detected by Western-blotting using appropriate antibodies. The full-length blots are presented in supplementary figures S18 , S19 .

Article Snippet: SB-202190 (#BML-EI294-001), PD-184352 (#ALX-270-471) were purchased from Alexis Biochemicals, CA, USA.

Techniques: Activation Assay, Incubation, Immunoprecipitation, Western Blot, Multiple Displacement Amplification, Inhibition

Journal: Respiratory Research

Article Title: Epithelial cell senescence impairs repair process and exacerbates inflammation after airway injury

doi: 10.1186/1465-9921-12-78

Figure Lengend Snippet: P38 MAPK inhibitor suppresses senescence-associated airway inflammation in mice . ( A ) Representative photomicrographs of double immunofluorescence staining for phospho-p38 MAPK ( green ) and CC10 ( red ) in the distal airway. Arrows indicate CC10-positive Clara cells that express phospho-p38 MAPK in their nuclei. Treatment with SB202190 of mice repeatedly exposed to NA and injected with BrdU reduces the proportion of Clara cells that express phospho-p38 MAPK ( B ) and the numbers of CD45-positive cells and CD90.2-positive cells infiltrating the distal airways ( C ) but does not affect the number of Clara cells ( D ) or the number of Clara cells that express p21 ( E ). Data are expressed as the means ± SEM. N = 4-6 in each experiment.

Article Snippet: The p38 mitogen-activated protein kinase (MAPK) inhibitor SB202190 (Enzo Life Sciences, Plymouth Meeting, PA) or 0.1% DMSO was administered by intraperitoneal injection 30 minutes before each BrdU injection.

Techniques: Mouse Assay, Double Immunofluorescence Staining, Injection

Journal: Respiratory Research

Article Title: Epithelial cell senescence impairs repair process and exacerbates inflammation after airway injury

doi: 10.1186/1465-9921-12-78

Figure Lengend Snippet: P38 MAPK activation in senescent Clara cells in the airways of COPD patients . ( A ) Representative photomicrographs of triple immunofluorescence staining of lung tissue sections obtained from COPD patients. Arrows indicate CC10-positive Clara cells that express both p16 and phospho-p38 MAPK. The arrowhead indicates a CC10-positive cell that expresses p16 but not phospho-p38 MAPK ( B ) Percentages of CC10-positive Clara cells that express p16, CC10-positive Clara cells that express phospho-p38 MAPK, and CC10-positive Clara cells that express both p16 and phospho-p38 MAPK in the lungs of COPD patients ( C : n = 14), asymptomatic smokers ( S : n = 7), and asymptomatic nonsmokers ( NS : n = 8). ( C ) Correlation between the percentage of CC10-positive Clara cells that express p16 and the percentage of CC10-positive Clara cells that express phospho-p38 MAPK. Open circles = asymptomatic nonsmokers; open triangles = asymptomatic smokers; closed circles = COPD patients. ( D ) Rates of immunopositivity for phospho-p38 MAPK in CC10-positive Clara cells that express p16 (senescent Clara cells) and in CC10-positive Clara cells that do not express p16 (presenescent Clara cells) in the subjects as a whole, asymptomatic nonsmokers, asymptomatic smokers, and COPD patients.

Article Snippet: The p38 mitogen-activated protein kinase (MAPK) inhibitor SB202190 (Enzo Life Sciences, Plymouth Meeting, PA) or 0.1% DMSO was administered by intraperitoneal injection 30 minutes before each BrdU injection.

Techniques: Activation Assay, Immunofluorescence, Staining

Journal: Respiratory Research

Article Title: Epithelial cell senescence impairs repair process and exacerbates inflammation after airway injury

doi: 10.1186/1465-9921-12-78

Figure Lengend Snippet: Senescence of NCI-H441 cells after exposure to BrdU for 10 days is accompanied by p38 MAPK-dependent pro-inflammatory cytokine production . ( A ) ELISA to measure concentrations of IL-6, TNF-α, and GM-CSF in the culture supernatants of NCI-H441 cells exposed or not exposed to 25 μM of BrdU in the presence or absence of 10 μM of the p38 MAPK inhibitor SB202190. The concentration of the anti-inflammatory cytokine IL-10 was below the limit of detection. ( B ) Immunoblot analyses for phosphorylation levels of p38 MAPK and NF-κB in the cell lysates. ( C ) Effects of SB202190 on BrdU-induced SA β-gal activation and growth arrest. Data are expressed as the means ± SEM. N = 3-6 in each experiment.

Article Snippet: The p38 mitogen-activated protein kinase (MAPK) inhibitor SB202190 (Enzo Life Sciences, Plymouth Meeting, PA) or 0.1% DMSO was administered by intraperitoneal injection 30 minutes before each BrdU injection.

Techniques: Enzyme-linked Immunosorbent Assay, Concentration Assay, Activation Assay

Journal: Respiratory Research

Article Title: Epithelial cell senescence impairs repair process and exacerbates inflammation after airway injury

doi: 10.1186/1465-9921-12-78

Figure Lengend Snippet: Pathways by which BrdU impairs epithelial repair and induces persistent inflammation in the chronic NA injury model . BrdU induces genotoxic stress, which activates the DNA damage response, thereby promoting premature senescence, which results in the growth arrest of epithelial cells. Genotoxic stress caused by BrdU also activates p38-MAPK pathways that trigger the production of pro-inflammatory cytokines/chemokines, which exacerbate inflammation. Which is necessary for p38-MAPK activation, the DNA damage response or cell cycle arrest (p21, etc.), has not been determined ( broken arrows ). Recent evidence indicates that pro-inflammatory cytokines (e.g., IL-6, IL-8) at least in part reinforce cell cycle arrest via the DNA damage response pathway [ 32 , 33 ], suggesting a positive feedback loop ( dashed arrow ) in which inflammation in turn promotes senescence.

Article Snippet: The p38 mitogen-activated protein kinase (MAPK) inhibitor SB202190 (Enzo Life Sciences, Plymouth Meeting, PA) or 0.1% DMSO was administered by intraperitoneal injection 30 minutes before each BrdU injection.

Techniques: Activation Assay

Journal: Mediators of Inflammation

Article Title: Luteolin Attenuates IL-1β-Induced THP-1 Adhesion to ARPE-19 Cells via Suppression of NF-κB and MAPK Pathways

doi: 10.1155/2020/9421340

Figure Lengend Snippet: Luteolin blocked P38 phosphorylation, and the P38 inhibitor SB202190 (SB) decreased THP-1 cell adherence to IL-1 β -stimulated ARPE-19 cells. (a) Western blots show levels of phosphorylated P38 protein expression. (b) The fold-change in pP38 protein expression was measured relative to P38 expression. (c) ARPE-19 cells were pretreated with 10 μ M luteolin or P38 inhibitor (SB203580) for 1 h and then cocultured with labeled THP-1 cells. (d) The fluorescence intensity was used to quantify calcein-AM fluorescence. Data represent the mean ± SD. ∗ p

Article Snippet: The inhibitors PD98059, SP600125, SB202190, and Bay 117082 were purchased from Enzo Life Sciences (Farmingdale, NY, USA).

Techniques: Western Blot, Expressing, Labeling, Fluorescence