Journal: The Journal of Cell Biology

Article Title: Hic-5 promotes invadopodia formation and invasion during TGF-β–induced epithelial–mesenchymal transition

doi: 10.1083/jcb.201108143

Figure Lengend Snippet: Matrix degradation by TGF-β–treated MCF10A cells or unstimulated GFP–Hic-5 MCF10A cells is dependent on p38 MAPK activity but not ERK. (A) TGF-β–treated MCF10A GFP cells were treated with control and hHic-5 siRNAs (hHic-5). Endogenous hHic-5 depletion reduces the level of p38 MAPK phosphorylation but not SMAD3 phosphorylation. (B) TGF-β–treated MCF10A GFP cells were plated on fluorescent 488–gelatin in the presence of vehicle, MEK/ERK inhibitor U0126, or p38 MAPK inhibitor SB203580. The p38 MAPK inhibitor blocked degradation, whereas the ERK inhibitor had no effect. Bar, 20 µm. (C) Quantitation of TGF-β–treated cells degrading matrix in the presence of inhibitors. (D) Western blot analysis showing that unstimulated GFP–Hic-5 cells have elevated levels of p38 MAPK phosphorylation. (E) GFP–Hic-5 cells plated on fluorescent gelatin in the presence of p38 MAPK and ERK inhibitors. P38 MAPK inhibition blocked matrix degradation. Bar, 20 µm. (F) Quantitation of GFP–Hic-5–expressing cells degrading matrix in the presence of inhibitors. (G) Invasion through Matrigel is reduced in GFP–Hic-5–expressing MCF10A cells by the p38 MAPK inhibitor, but not by ERK inhibition. ***, P < 0.0005. (H) Treatment of GFP–Hic-5 cells with Rac1 inhibitor NSC23766 reduces the level of phospho-p38 MAPK. Molecular mass standards are indicated next to the gel blots in kilodaltons. Error bars represent the standard error of the mean.

Article Snippet: Inhibitors were used as follows: 2 μM of Src inhibitor PP2, 10 μM of Rac1 inhibitor NSC23766, 10 μM of ROCK inhibitor Y27632, 10 μM of p38 MAPK inhibitor SB203580, and 10 μM of MEK/ERK inhibitor U0126 were from EMD; and 2 μM of FAK inhibitor PF573228 was purchased from Tocris Bioscience.

Techniques: Activity Assay, Control, Phospho-proteomics, Quantitation Assay, Western Blot, Inhibition, Expressing

Journal: The Journal of Cell Biology

Article Title: Hic-5 promotes invadopodia formation and invasion during TGF-β–induced epithelial–mesenchymal transition

doi: 10.1083/jcb.201108143

Figure Lengend Snippet: Hic-5 up-regulation during TGF-β–stimulated EMT promotes the development of an invasive phenotype through activation of multiple signaling pathways. TGF-β treatment results in the increased expression of Hic-5. Src is phosphorylated in response to Hic-5 expression, and Src activity is necessary for the phosphorylation of Hic-5, resulting in a positive feedback activation of Src. TGF-β–stimulated ROCK-mediated matrix degradation is downstream of Hic-5 expression and requires RhoC, but not RhoA. Hic-5 up-regulation also stimulates the activation of Rac1–p38 MAPK, which is also required for the invasive phenotype including invadopodia formation, matrix degradation, and increased invasion.

Article Snippet: Inhibitors were used as follows: 2 μM of Src inhibitor PP2, 10 μM of Rac1 inhibitor NSC23766, 10 μM of ROCK inhibitor Y27632, 10 μM of p38 MAPK inhibitor SB203580, and 10 μM of MEK/ERK inhibitor U0126 were from EMD; and 2 μM of FAK inhibitor PF573228 was purchased from Tocris Bioscience.

Techniques: Activation Assay, Protein-Protein interactions, Expressing, Activity Assay, Phospho-proteomics

Journal: Journal of Biological Chemistry

Article Title: Signaling Events Involved in Interleukin 27 (IL-27)-induced Proliferation of Human Naive CD4+ T Cells and B Cells

doi: 10.1074/jbc.m111.221010

Figure Lengend Snippet: FIGURE 6. Analysis of the role of MAPK in IL-27-mediated proliferation of naive CD4 T cells. A, purified naive CD4 T cells were cultured for 4 days with plate-coated CD3 mAb with or without IL-27, in the absence (control) or presence of U0126, SP600125 (SP600), or SB203580 (SB203) (all at 10 M). Proliferation was measured by [3H]thymidine incorporation. Results are expressed in cpm (mean of triplicates S.D.) and are representative of four independent experi- ments performed with 3 to 4 different donors. B, increase in cpm induced by IL-27 in the presence of the inhibitors relative to that observed in its absence is shown (mean S.E. of four experiments). C, CFSE-labeled naive and memory CD4 T cells were cultured as described in A and analyzed by FACS to monitor cell division. The percentage of cells that underwent over 1 division and its increase induced by IL-27 are indicated.

Article Snippet: The following chemical inhibitors were used: U0126, SB203580, and SP600125 (Calbiochem), Pim-1 inhibitor 2 (Santa Cruz Biotechnology) and c-Myc inhibitor (10058-F4, Sigma-Aldrich).

Techniques: Purification, Cell Culture, Control, Labeling

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: Investigative ophthalmology & visual science

Article Title: Transforming Growth Factor-β1-induced Human Subconjunctival Fibrosis is Mediated by MicroRNA 143/145 Expression.

doi: 10.1167/iovs.19-26797

Figure Lengend Snippet: FIGURE 3. Role of p38MAPK, JNK, ERK1/2, PI3K, Akt, SMAD2, and SMAD4 in TGF-b1–induced miRNA expression in human Tenon’s capsule fibroblast. (A–C) Inhibitors of p38MAPK (SB203580), JNK (SP600125), ERK1/2 (U0126), PI3K (LY294002), and Akt (SH5) revealed decreased expression of TGF-b1–induced miRNA 143/145. (D) However, treatment of siRNAs to SMAD2 and SMAD4 did not affect TGF-b1–induced miRNA 143/145 expression.

Article Snippet: SB203580 (specific inhibitor of p38MAPK), LY294002 (inhibitor of PI3K), and U0126 (inhibitor of ERK1/2) were from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Expressing

Journal: Biophysical Journal

Article Title: Fluid Shear Stress Increases Neutrophil Activation via Platelet-Activating Factor

doi: 10.1016/j.bpj.2014.04.001

Figure Lengend Snippet: Shear- and PAF-induced L-selectin shedding is ADAM 17- and p38 MAP kinase-dependent. L-selectin expression as a function of forward scatter of nonsheared and sheared (1.0 dyn/cm 2 ) neutrophils in the absence of PAF ( A and B ) after treatment with PAF ( C and D ), and PAF-stimulated cells treated with TAPI-0 ( E and F ) and SB203580 ( G and H ). Gate determined using fluorescence of isotype controls. ( I ) Quantification of L-selectin shedding in all samples. n = 3 separate donors. ∗ P < 0.05. NS , not significant. To see this figure in color, go online.

Article Snippet: Tumor necrosis factor- α protease inhibitor-0 (TAPI-0) and p38 mitogen-activated protein (MAP) kinase inhibitor SB203580 were purchased from Peptides International (Louisville, Kentucky) and Millipore, respectively.

Techniques: Expressing, Fluorescence