Journal: Evidence-based Complementary and Alternative Medicine : eCAM

Article Title: A Taiwanese Propolis Derivative Induces Apoptosis through Inducing Endoplasmic Reticular Stress and Activating Transcription Factor-3 in Human Hepatoma Cells

doi: 10.1155/2013/658370

Figure Lengend Snippet: The propolis derivative, GS-002, induced ATF-3 expression that was mediated by MAPK pathways. Hep3B cells were treated with (a) 20 μ g/mL GS-002 for the indicated time periods, or (b) with various concentrations of GS-002 for 1 h. Total cell lysates were used to detect protein expressions of p38, phosphor-p38 (p-p38), c-Jun N-terminal kinase (JNK), phospho-JNK (p-JNK), extracellular signal-regulated kinase (ERK), and phospho-ERK (p-ERK) by Western blotting. Ten micrograms of 15-deoxy-Δ12,14-prostaglandin J 2 (15d-PGJ 2 ) was used as a positive control. (c) Hep3B cells were pretreated with 5 and 10 μ M of the p38 inhibitor, SB203580, the ERK inhibitor, PD98059, or the JNK inhibitor, SP600125, for 90 min, then treated with GS-002 (20 μ g/mL) for another 12 h, and the ATF-3 protein level was detected by Western blotting.

Article Snippet: Inhibitors of SP600125, SB203580, and PD98059 were purchased from Tocris Bioscience (Bristol, UK).

Techniques: Expressing, Western Blot, Positive Control

Journal: Journal of Inflammation Research

Article Title: Usp18 Mediates D-Dopachrome Tautomerase-Induced Astrocytic Inflammation After Spinal Cord Injury

doi: 10.2147/jir.s505433

Figure Lengend Snippet: Figure 7 Determination of Usp18 expression in the astrocytes following treatment with MAPKs inhibitors. (a) Western blot analysis of protein levels of phosphorylated ERK, JNK, P38 kinase and Usp18 after astrocyte treatment with 10 μM P38 (SB203580), ERK (PD98059) or JNK (SP600125) inhibitor in the presence of 1 μg/mL rD-DT for 24h. Quantities were normalized to endogenous GAPDH. (b) Quantification data as shown in (a). Error bars represent the SEM (*P < 0.05). (c) RT-PCR analysis of Usp18 expression in the astrocytes with the same treatment. Experiments were performed at least in triplicates. Error bars represent the SEM (*P < 0.05).

Article Snippet: To examine the effects of D-DT on the expression of Usp18, the primary astrocytes were treated with various selective inhibitor SB203580 (TOCRIS), SP600125 (TOCRIS) or PD98059 (TOCRIS) in the presence or absence of 1 μg/mL recombinant D-DT (Aviva Systems Biology) for 24 h prior to biochemical assay.

Techniques: Expressing, Western Blot, Reverse Transcription Polymerase Chain Reaction

Journal: The Journal of biological chemistry

Article Title: p38 mitogen-activated protein kinase negatively regulates the induction of phase II drug-metabolizing enzymes that detoxify carcinogens.

doi: 10.1074/jbc.275.4.2322

Figure Lengend Snippet: FIG. 1. Inhibition of tBHQ-induced p38 activation by SB203580. HepG2 (A) or Hepa1c1c7 (B) cells were incubated with SB203580 (5 mM), SB202474 (5 mM), or solvent (0.1% Me2SO) for 1 h prior to challenge with tBHQ (100 mM) for an additional 1 h. Cells were harvested, and the endogenous p38 kinase activity was determined by immunocomplex kinase assays using 5 mg of GST-ATF2-(1–96) fusion protein as substrate. The protein level of p38 was determined by West- ern blotting. The experiment was repeated three times.

Article Snippet: The specific p38 inhibitor, SB203580, and its negative analog, SB202474, were purchased from Calbiochem. tBHQ, BHA, and b-NF were purchased from Aldrich, and SUL was purchased from LKT Laboratories (St. Paul, MN).

Techniques: Inhibition, Activation Assay, Incubation, Solvent, Activity Assay

Journal: The Journal of biological chemistry

Article Title: p38 mitogen-activated protein kinase negatively regulates the induction of phase II drug-metabolizing enzymes that detoxify carcinogens.

doi: 10.1074/jbc.275.4.2322

Figure Lengend Snippet: FIG. 2. Enhancement of tBHQ-induced QR activity by SB203580. Hepa1c1c7 cells were treated with SB203580 (5 mM), SB202474 (5 mM), or solvent (0.1% Me2SO) for 1 h and then stimulated with tBHQ (100 mM) for 24 h. Cells were harvested and assayed for QR activity by measuring the reduction of 2,6-dichloroindophenol as de- scribed under “Materials and Methods.” The data shown are means of four independent experiments 6 S.D.

Article Snippet: The specific p38 inhibitor, SB203580, and its negative analog, SB202474, were purchased from Calbiochem. tBHQ, BHA, and b-NF were purchased from Aldrich, and SUL was purchased from LKT Laboratories (St. Paul, MN).

Techniques: Activity Assay, Solvent

Journal: The Journal of biological chemistry

Article Title: p38 mitogen-activated protein kinase negatively regulates the induction of phase II drug-metabolizing enzymes that detoxify carcinogens.

doi: 10.1074/jbc.275.4.2322

Figure Lengend Snippet: FIG. 3. SB203580 potentiates the induction of ARE-luciferase reporter gene by tBHQ. A, HepG2 cells were transiently transfected with 0.5 mg of pCH110-b-gal plasmid and 1.5 mg of ARE-TI-luciferase reporter construct or the construct without ARE enhancer (TI-Luc). After transfection, cells were incubated for 12 h in culture medium and then incubated with SB203580 (5 mM) or SB202474 (5 mM) for 1 h, prior to treatment with tBHQ (100 mM) for 24 h. Luciferase activity was determined and normalized against b-galactosidase activity. The amount of luciferase activity in the cells that were transfected with ARE-luciferase construct but treated with solvent alone was given an arbitrary value of 1. B, HepG2 cells were transfected with an ARE- luciferase reporter construct, and stably transfected cell clones were selected as described under “Materials and Methods.” C4, a randomly selected positive clone, was treated with the indicated concentrations of SB203580 or SB202474 for 1 h before the addition of tBHQ (100 mM). Luciferase activity was determined 24 h after treatment and normal- ized against protein concentration. The level of luciferase activity in untreated C4 cells was arbitrarily set to 1. The data shown are means of three independent experiments performed in duplicate.

Article Snippet: The specific p38 inhibitor, SB203580, and its negative analog, SB202474, were purchased from Calbiochem. tBHQ, BHA, and b-NF were purchased from Aldrich, and SUL was purchased from LKT Laboratories (St. Paul, MN).

Techniques: Luciferase, Transfection, Plasmid Preparation, Construct, Incubation, Activity Assay, Solvent, Stable Transfection, Clone Assay, Protein Concentration

Journal: The Journal of biological chemistry

Article Title: p38 mitogen-activated protein kinase negatively regulates the induction of phase II drug-metabolizing enzymes that detoxify carcinogens.

doi: 10.1074/jbc.275.4.2322

Figure Lengend Snippet: FIG. 5. Effects of BHA, b-NF, and SUL on ARE-mediated gene expression and p38 activity. A, augmentation of BHA, b-NF, and SUL induction of ARE-mediated reporter gene activity by SB203580. Stably transfected C4 cells were pretreated with SB203580 (5 mM), SB202474 (5 mM), or solvent (0.1% Me2SO) for 1 h prior to the addition of BHA (100 mM), b-NF (5 mM), SUL (12.5 mM), or solvent. Luciferase activity was determined 24 h after drug treatment and normalized by protein concentration. The data, as expressed as -fold induction over control (solvent-treated cells), are means and S.D. values of four inde- pendent experiments. B, activation of p38 by BHA, b-NF, and SUL. C4 cells were untreated or pretreated with SB203580 (5 mM) before incu- bation with BHA (100 mM), b-NF (5 mM), or SUL (12.5 mM) for 1 h. The endogenous p38 activity was immunoprecipitated with a specific anti- body and assayed with GST-ATF2-(1–96) fusion protein as substrate. The protein level of p38 was determined by Western blotting. The experiment was repeated twice.

Article Snippet: The specific p38 inhibitor, SB203580, and its negative analog, SB202474, were purchased from Calbiochem. tBHQ, BHA, and b-NF were purchased from Aldrich, and SUL was purchased from LKT Laboratories (St. Paul, MN).

Techniques: Gene Expression, Activity Assay, Stable Transfection, Transfection, Solvent, Luciferase, Protein Concentration, Control, Activation Assay, Immunoprecipitation, Western Blot

Journal: The Journal of biological chemistry

Article Title: p38 mitogen-activated protein kinase negatively regulates the induction of phase II drug-metabolizing enzymes that detoxify carcinogens.

doi: 10.1074/jbc.275.4.2322

Figure Lengend Snippet: FIG. 6. SB203580 induces an earlier kinetics of ARE-dependent gene activation than tBHQ. C4 cells were treated with SB203580 (5 mM) or tBHQ (100 mM) for different times. Luciferase activity was determined and normalized by protein concentration. The data ob- tained from three separate experiments were expressed as -fold induc- tion over control (solvent-treated cells).

Article Snippet: The specific p38 inhibitor, SB203580, and its negative analog, SB202474, were purchased from Calbiochem. tBHQ, BHA, and b-NF were purchased from Aldrich, and SUL was purchased from LKT Laboratories (St. Paul, MN).

Techniques: Activation Assay, Luciferase, Activity Assay, Protein Concentration, Control, Solvent

Journal: Frontiers in Immunology

Article Title: Targeting the oxidative stress-neuroinflammation axis: the mechanism of arctigenin’s broad-spectrum analgesia with limited side effects

doi: 10.3389/fimmu.2026.1754756

Figure Lengend Snippet: The antiallodynic effects of AG in SNI mice are associated with MAPK signaling pathway. (A) A shows western blotting of p-ERK and t-ERK in each group of spinal cord and qualitative data of protein expression in different groups. (B) Western blotting of p-JNK and t-JNK in different groups. (C) Western blotting of p-p38 and t-p38 in different groups. (D) Western blotting of p-mTOR in different groups. (E) Western blotting of PGC-a in different groups. (F) Western blotting of p-AMPK in different groups. (G) The analgesic effects of AG in SNI mice could be blocked by pretreatment with inhibitors of the MAPK pathway [U0126 (100 μg/kg, i.p.), SB203580 (40 μg/ml, 10 ml/kg, i.p.), or SP600125 (50 μg/kg, i.p.)], as measured by von Frey testing. (H) The extent and duration of analgesia are estimated by the area under curve (AUC (g min)) of PWT vs time (0-120 minutes). Data are expressed as the mean ± SEM. ** p < 0.01 and *** p < 0.001 for sham group vs SNI group. # p < 0.05, and ## p < 0.01 for SNI group vs AG+SNI group. && p < 0.01 compared with AG+SNI group. The "ns" indicates no significant difference.

Article Snippet: U0126 (inhibitor of ERK, cat number: HY-12031A), SB20358 (inhibitor of p38, cat number: HY-112349) and SP600125 (inhibitor of JNK, cat number: HY-12041) were purchased from MedChemExpress.

Techniques: Western Blot, Expressing

Journal: Oncogene

Article Title: A chemical inhibitor of PPM1D that selectively kills cells overexpressing PPM1D.

doi: 10.1038/sj.onc.1210729

Figure Lengend Snippet: Figure 1 Reduction of high-level PPM1D expression can selectively inhibit the growth of human tumour cells. (a) Validation of pSUPER-mediated RNAi in cells overexpressing PPM1D. 293T cells were transfected with a construct expressing full-length PPM1D fused to enhanced green fluorescent protein (eGFP) and either pSUPER-PPM1D (lane A) or pSUPER-control (lane B). Lane C represents cells transfected with the PPM1D-eGFP fusion construct but no pSUPER construct. Cell lysates were generated and western blotted 48 h after transfection. The top panel shows lysates probed with an anti-GFP antibody and in the bottom panel, the same western blot is shown probed with an anti-b-tubulin antibody as a loading control. (b) Colony formation in human tumour cell lines after inhibition of PPM1D expression using pSUPER-PPM1D. Cells were transfected with either pSUPER-PPM1D or pSUPER- control and also a plasmid encoding blasticidin resistance. After 14 days of blasticidin selection, colonies were quantified. (c) Quantification of colony formation in a range of human tumour cell lines after PPM1D reduction. The surviving fraction (SF) was calculated for each cell line using the equation SF (%) ¼ (number of colonies from cells transfected with pSUPER-PPM1D/number of colonies from cells transfected with pSUPER-control) 100. Each bar represents three independent experiments and the error bars, two standard errors of the mean. (d) PPM1D and P38 expression profiles of human tumour cell lines by western blot analysis. (e) P38 inhibition rescues PPM1D RNAi silencing-mediated cell death. MCF-7 cells were treated with SB203580 (10 mM) for 24 h before transfection with either pSUPER-control or pSUPER-PPM1D. SB203580 (10 mM) results in a 50% inhibition of P38 phosphorylation of ATF2 (data not shown). Use of SB203580 at >10 mM inhibited the transfection procedure (data not shown). Cells were retreated with SB203580 24 h after transfection. Cells were counted at 48 h post-transfection and the SF was calculated for each transfection (according to the number of cells originally plated). Each bar represents three independent experiments and the error bars, two standard errors of the mean.

Article Snippet: To investigate the mechanism of cell death upon PPM1D RNAi silencing, we examined the effects of treating MCF-7 cells with the specific P38 inhibitor SB203580 (Fitzgerald et al., 2003).

Techniques: Expressing, Biomarker Discovery, Transfection, Construct, Control, Generated, Western Blot, Inhibition, Plasmid Preparation, Selection, Phospho-proteomics

Journal: Oncogene

Article Title: A chemical inhibitor of PPM1D that selectively kills cells overexpressing PPM1D.

doi: 10.1038/sj.onc.1210729

Figure Lengend Snippet: Figure 3 Effect of chemical inhibitors on PPM1D phosphatase activity. Western blots are shown detailing for each compound the effect of increasing concentration (10–50 mM) of inhibitors on phosphorylation of full-length recombinant P38 (at approximately 50 kDa) in the presence of rPPM1D and MgCl2 in an in vitro assay. In each case, total P38 was used as a loading control.

Article Snippet: To investigate the mechanism of cell death upon PPM1D RNAi silencing, we examined the effects of treating MCF-7 cells with the specific P38 inhibitor SB203580 (Fitzgerald et al., 2003).

Techniques: Activity Assay, Western Blot, Concentration Assay, Phospho-proteomics, Recombinant, In Vitro, Control

Journal: Oncogene

Article Title: A chemical inhibitor of PPM1D that selectively kills cells overexpressing PPM1D.

doi: 10.1038/sj.onc.1210729

Figure Lengend Snippet: Figure 4 Effect of PPM1D inhibitors on cell viability. (a) The effect of CCT007093 and SB203580 (10 mM) on MCF-7 and HeLa cells. Cells were treated with SB203580 on days 1, 2 and 3 post- plating. Cells were also treated with CCT007093 on day 2. Viable cells were counted on day 4 and the surviving fraction calculated. (b) P38 phosphorylation in cells treated with CC000913 or CCT007093. Cells were treated with CC000913 or CCT007093 and lysates collected at the time points shown. Western blotting to detect pP38, total P38 and b-tubulin was performed as shown. H2O2 (1 mM) and dimethyl sulphoxide (DMSO) were used as the positive and negative controls respectively.

Article Snippet: To investigate the mechanism of cell death upon PPM1D RNAi silencing, we examined the effects of treating MCF-7 cells with the specific P38 inhibitor SB203580 (Fitzgerald et al., 2003).

Techniques: Phospho-proteomics, Western Blot