Journal: Frontiers in Oncology

Article Title: Cytotoxic Effects of Arsenite in Combination With Gamabufotalin Against Human Glioblastoma Cell Lines

doi: 10.3389/fonc.2021.628914

Figure Lengend Snippet: Prosurvival role of p38 MAPK in the cytotoxicity of U-87 cells treated with the combination of As III and gamabufotalin. (A) Following treatment for 48 h with relatively low concentrations of As III (1, 2 μM) and gamabufotalin (20, 50 nM), alone or in combination, the expression profiles of phospho-p38 (p-p38) and p38 were analyzed using western blotting. A representation image of the expression profile of each protein is shown from three independent experiments. The expression levels were expressed as the ratios between each targeted protein and β-actin protein expression levels, and were compared with those of control group. (B) Following treatment for 48 h with the combined regimen of 2 μM As III + 50 nM gamabufotalin; 3.3 μM As III + 40 nM gamabufotalin, in the presence of absence of 5 μM SB203580, a specific inhibitor for p38 MAPK and its negative control SB202474, cell viability was determined by XTT assay. Relative cell viability was calculated as the ratio of the absorbance at 450 nm of each treatment group against those of the corresponding untreated control group. Data are shown as the means ± SD (n ≥ 3). A p value less than 0.05 was considered as statistically significant ( § p < 0.001; ‡ p < 0.0001 vs. control. $ p < 0.001 vs. As+Gama and As+Gama+SB202474). As, As III ; Gama, gamabufotalin; SB203, SB203580; SB202, SB202474. The images of beta-actin are identical to that in Figure 4B since the same experiment samples were used to analyze.

Article Snippet: Both SB203580, a specific inhibitor of p38 MAPK, and its negative control SB202474 were purchased from Merck KGaA.

Techniques: Expressing, Western Blot, Negative Control, XTT Assay

Journal: Scientific Reports

Article Title: Angiotensin-converting enzyme 2 prevents lipopolysaccharide-induced rat acute lung injury via suppressing the ERK1/2 and NF-κB signaling pathways

doi: 10.1038/srep27911

Figure Lengend Snippet: LPS exposure caused a marked increase of the phosphorylation levels of p38 MAPK ( A ), ERK1/2 ( B ) and JNK ( C ), which was significantly suppressed by ACE2 overexpression in rat lung. Pretreatment with A779 or MLN-4760 completely abolished the inhibitory effects of ACE2 overexpression on LPS-induced p38 MAPK and ERK1/2 phosphorylation, but did not affect the level of JNK phosphorylation. ACE2 RNAi in rat lung significantly enhanced the LPS-induced ERK1/2 and JNK phosphorylation but did not change p38 MAPK phosphorylation. Data are represented as mean ± SD. *p < 0.05, versus control group; # p < 0.05, versus LPS group; $ p < 0.05, versus ACE2 group (n = 6, per group).

Article Snippet: SB203580 (a specific inhibitor of p38 MAPK), PD98059 (a specific inhibitor of ERK1/2) and SP600125 (a specific inhibitor of JNK) were purchased from Santa Cruz Biotechnology (Delaware, CA, USA).

Techniques: Phospho-proteomics, Over Expression, Control

Journal: Scientific Reports

Article Title: Angiotensin-converting enzyme 2 prevents lipopolysaccharide-induced rat acute lung injury via suppressing the ERK1/2 and NF-κB signaling pathways

doi: 10.1038/srep27911

Figure Lengend Snippet: In LPS+ACE2-RNAi group, SB203580, PD98059 or SP600125 pretreatment significantly suppressed LPS-induced TNF-α and IL-1β secretion in rat BLAF. In Lenti- Ace2 infected rats, pretreatment with SB203580, PD98059 or SP600125 reversed the blockade effects of A779 or MLN4760 on ACE2 overexpression, and markedly attenuated LPS-induced cytokine secretion in BALF. Data are represented as mean ± SD. *p < 0.05, versus control group; # p < 0.05, versus LPS group; $ p < 0.05, versus ( A , D ) LPS+ACE2-RNAi group, ( B , E ) LPS+ACE2+A779 group, ( C , F ) LPS+ACE2+MLN4760 group (n = 6, per group).

Article Snippet: SB203580 (a specific inhibitor of p38 MAPK), PD98059 (a specific inhibitor of ERK1/2) and SP600125 (a specific inhibitor of JNK) were purchased from Santa Cruz Biotechnology (Delaware, CA, USA).

Techniques: Infection, Over Expression, Control

Journal: Journal of Biomedical Science

Article Title: Suberoylanilide hydroxamic acid represses glioma stem-like cells

doi: 10.1186/s12929-016-0296-6

Figure Lengend Snippet: SAHA induces GSCs senescence via activation of the p38-p53 pathway. a to c GSCs from U87MG cells were collected 7 days after treatment of SAHA at different doses. The cellular lysates were then analyzed by Western blotting with indicated antibodies ( a ). The senescence-associated β-galactosidase (SA-β-gal) activities were then examined by SA-β-gal kit (Cell Signaling, details described in materials and methods). Photomicrographs of spheroid bodies were randomly selected in microscopic fields ( b ) and the percentage of SA-β-gal positive cells was quantified; bar, S.D.; ** p < 0.01 ( c ). d to g GSCs were pre-treated with or without 3 μM SB203580 (SB) for 1 h and then treated with DMSO or with SAHA (1 μM in G, 2.5 μM in d to g ) for 7 days. After treatment, cellular senescence was measured by the detection of SA-β-gal activity ( d ). The percentage of SA-β-gal positive cells was quantified; bar, S.D., *, P < 0.05, **, P < 0.01 ( e ). The protein lysates were analyzed by Western blotting with indicated antibodies ( f and g ). h GSCSs were treated with DMSO or with different doses of SAHA as indicated for 2 days. After treatment, the protein lysates were analyzed by Western blotting

Article Snippet: SB203580, a specific p38 inhibitor, was purchased from Selleckchem (S1076) and dissolved in DMSO.

Techniques: Activation Assay, Western Blot, Activity Assay

Journal: Journal of Biomedical Science

Article Title: Suberoylanilide hydroxamic acid represses glioma stem-like cells

doi: 10.1186/s12929-016-0296-6

Figure Lengend Snippet: A diagram illustrates the signaling mechanisms of how SAHA may be inhibiting GSCs. Low-dose of SAHA increases both p53 protein and its phosphorylation at Ser33 via p38 activation, leading to a rise in cell cycle checkpoint activation and cellular sensecence, but high-dose of SAHA induces p53 Ser15 phosphorylation and caspase-mediated involving both the caspase-8 and the caspase-9 pathways in GSCs

Article Snippet: SB203580, a specific p38 inhibitor, was purchased from Selleckchem (S1076) and dissolved in DMSO.

Techniques: Phospho-proteomics, Activation Assay

Journal: Journal of Cancer Research and Clinical Oncology

Article Title: Effects of VEGFR1 + hematopoietic progenitor cells on pre-metastatic niche formation and in vivo metastasis of breast cancer cells

doi: 10.1007/s00432-018-2802-6

Figure Lengend Snippet: HPCs enhances the invasive growth of MDA-MB-435s-HM cells. HPCs were co-cultured with MDA-MB-435s-HM cells, and cells were treated with an inhibitor of LY-294002, PD98059, SB431542, CP690550, or BIRB796. The invasive growth was assessed based on a Micro PET photographs, as area of nodules in liver formed by MDA-MB-435s-HM cells or Masson staining indicating the invasive growth of cells. Quantitative results from b Micro PET photographs; c fold difference in radioactivity between liver and blood; d inhibitory rates of inhibitors on 18 F-FDG intensity or e invasive growth of cells. Definitions: inhibitor of MMP-9, tissue inhibitor of metalloproteinase-1 (TIMP-1) shown to be a natural inhibitor of MMP-9; anti-CD31 antibody, anti-human CD31 antibody recognizing the D2 extracellular portion of CD31 to block its function; inhibitor for PDGFR-α, PDGFR tyrosine kinase inhibitor III shown to be an ATP-competitive inhibitor of PDGFR-α. * P < 0.05. White arrow in Figure indicates the nodules in mouse liver

Article Snippet: Small molecular inhibitors, LY294002 (a specific inhibitor of PI3K/AKT pathway, cat #S1105), PD98059 (a specific inhibitor of MEK/ERK pathway, cat #S1177), CP690550 (a specific inhibitor of Jak/STAT pathway, cat #S2789), SB431542 (a specific inhibitor of TGFβ/Smad pathway, cat #S1067), and BIRB796 (a specific inhibitor of p38 MAPK pathway, cat #S1574) were purchased from Selleck Corporation (Houston, TX, USA).

Techniques: Cell Culture, Micro-PET, Staining, Radioactivity, Blocking Assay

Journal: Journal of Biological Chemistry

Article Title: Active Mutants of the Human p38α Mitogen-activated Protein Kinase

doi: 10.1074/jbc.m404595200

Figure Lengend Snippet: FIG. 1. Multiple sequence alignment (ClustalW and EMBL) of several members of the MAP kinase family. The sequences include relevant segments of the proteins and are from human sources except for Hog1 (yeast). The original sites of activating mutants identified by the genetic screen in Hog1 are marked with short black arrows. The conserved residues between Hog1 and p38 are bounded within blocks. Amino acids with chemical properties similar to those of the mutated residues are marked in boldfaced type. Asn-391 of Hog1, one of the selected mutation sites, is not included in this sequence alignment because it is located in a C-terminal region, which is lacking in all p38 proteins.

Article Snippet: The WT and active mutants of p38 were subjected to a Western blot using a specific antiphospho-p38 (Cell Signaling, catalog number 9211) before and after MKK6 treatment (lower image).

Techniques: Sequencing, Mutagenesis

Journal: Journal of Biological Chemistry

Article Title: Active Mutants of the Human p38α Mitogen-activated Protein Kinase

doi: 10.1074/jbc.m404595200

Figure Lengend Snippet: FIG. 2. Some of the p38 mutants are catalytically active. The mutants, purified from E. coli, were subjected to a kinase assay with (MKK6) or without (MKK6) active MKK6 phosphorylation using GST-ATF2 as a substrate. A, a fixed volume from each reaction was loaded on the gel. Coomassie staining verified that equal amounts of substrate were loaded (upper images). Equal exposures were applied for the detection of radioactivity in both gels (lower images). The p38D176A, p38F327L and p38F327S variants are active independently of MKK6 phos- phorylation. Furthermore, all of the assayed mutants retained their propensity to be further activated by MKK6. B, using the paper-spotted kinase assay technique, we quantified and normalized the activities of the mutants to that of the fully activated WT p38 that was defined as 100%. The graph shows averages of results from several independent experiments. The p38 mutants exhibited up to 10% activity relative to active WT p38. S.E. values are 4% for the unphosphorylated and phosphorylated variants. C, WT p38 and the two mutants were sub- jected to kinase assay and, subsequently, to SDS-PAGE. Coomassie staining (upper image) verified equal amounts of substrate in each lane. Equal exposures were applied for the detection of radioactivity (lower image). p38D179A is intrinsically active, whereas p38F330S remain inactive, similarly to unphosphorylated WT p38.

Article Snippet: The WT and active mutants of p38 were subjected to a Western blot using a specific antiphospho-p38 (Cell Signaling, catalog number 9211) before and after MKK6 treatment (lower image).

Techniques: Purification, Kinase Assay, Phospho-proteomics, Staining, Radioactivity, Activity Assay, SDS Page

Journal: Journal of Biological Chemistry

Article Title: Active Mutants of the Human p38α Mitogen-activated Protein Kinase

doi: 10.1074/jbc.m404595200

Figure Lengend Snippet: FIG. 3. Kinase activity of double and single mutants relative to the activity of MKK6-activated WT p38. The two variants p38D176A/F327L (D176AF327L) and p38D176A/F327S (D176AF327S) ex- hibited 25% activity relative to active WT p38 (WT), an increase of 2.5-fold in activity relative to the active single mutant p38F327S (F327S). This graph shows averages of results from several independent exper- iments. S.E. values are 4%.

Article Snippet: The WT and active mutants of p38 were subjected to a Western blot using a specific antiphospho-p38 (Cell Signaling, catalog number 9211) before and after MKK6 treatment (lower image).

Techniques: Activity Assay, Mutagenesis

Journal: Journal of Biological Chemistry

Article Title: Active Mutants of the Human p38α Mitogen-activated Protein Kinase

doi: 10.1074/jbc.m404595200

Figure Lengend Snippet: FIG. 4. Active mutants display lin- ear activity as a function of both time and enzyme concentrations and fol- low Michaelis-Menten kinetics. A, two active mutants, p38D176A (D176A) and p38D176A/F327S (D176AF327S), as well as non-activated WT protein were subjected to kinase assay using increasing time in- tervals and found to be linear at the en- tire measured time frame (up to 40 min). B, the same mutants as in panel A were subjected to kinase assay using increas- ing enzyme concentrations (up to 1 g per reaction well) and found to be linear. C, Lineweaver-Burk plots of the kinetic as- say performed on the double mutant p38D176A/F327S (D176AF327S) and the dually phosphorylated WT p38 (Activated WT). All kinetics profiles followed the Michaelis-Menten model. Using extrapo- lation, the kinetic parameters were deter- mined to be Vmax and Km values of 37 (picomoles of phosphate incorporated min1 milligrams of enzyme1) and 25.3 M, respectively, for p38D176A/F327S

Article Snippet: The WT and active mutants of p38 were subjected to a Western blot using a specific antiphospho-p38 (Cell Signaling, catalog number 9211) before and after MKK6 treatment (lower image).

Techniques: Activity Assay, Kinase Assay, Mutagenesis

Journal: Journal of Biological Chemistry

Article Title: Active Mutants of the Human p38α Mitogen-activated Protein Kinase

doi: 10.1074/jbc.m404595200

Figure Lengend Snippet: FIG. 5. p38 active mutants retained specificity toward sub- strates. A, an in vitro kinase assay was conducted with three different substrates, namely GST-ATF2, GST-c-Jun, and the myelin basic pro- tein (MBP) (Sigma M-1891). A fixed volume from each reaction was loaded on a gel. Coomassie staining (upper images) indicated that equal amounts of substrates were loaded. Short exposure (45 min without intensifier) of the gel to a film (short) revealed that p38D176A (D176A), p38F327S (F327S), p38D176A/F327L (D176AF327L), and p38D176AF327S

Article Snippet: The WT and active mutants of p38 were subjected to a Western blot using a specific antiphospho-p38 (Cell Signaling, catalog number 9211) before and after MKK6 treatment (lower image).

Techniques: In Vitro, Kinase Assay, Staining

Journal: Journal of Biological Chemistry

Article Title: Active Mutants of the Human p38α Mitogen-activated Protein Kinase

doi: 10.1074/jbc.m404595200

Figure Lengend Snippet: FIG. 6. p38 active mutants are inhibited by specific inhibitors. A, an in vitro kinase assay was conducted with GST-ATF2 as a sub- strate. The two most active mutants p38D176A/F327L (p38D176AF327L) and p38D176A/F327S (p38D176AF327S)were tested with increasing concentra- tions of SB-203580 and PD-169316. A fixed volume from each kinase reaction was loaded on a gel. Coomassie staining (upper image) verified that equal amounts of substrate were loaded. Radioactivity was re- vealed by exposure of the gel to a film (lower image). The two mutants are efficiently inhibited with both SB-203580 and PD-169316. 1 M PD-169316 completely blocked activity in both cases, whereas 1 M SB-203580 was not sufficient for a complete blockage. This result was expected, as PD-169316 is more potent inhibitor than SB-203580. B, the inhibition pattern of p38D176A/F327S (p38 D176AF327S) and of acti- vated WT p38 by SB-203580 is essentially similar. As expected, there is a 1:4 ratio in the level of the kinase activity between the unphospho- rylated active mutant and the dually phosphorylated WT p38.

Article Snippet: The WT and active mutants of p38 were subjected to a Western blot using a specific antiphospho-p38 (Cell Signaling, catalog number 9211) before and after MKK6 treatment (lower image).

Techniques: In Vitro, Kinase Assay, Staining, Radioactivity, Activity Assay, Inhibition, Mutagenesis

Journal: Journal of Biological Chemistry

Article Title: Active Mutants of the Human p38α Mitogen-activated Protein Kinase

doi: 10.1074/jbc.m404595200

Figure Lengend Snippet: FIG. 7. Western blot (WB) analysis of the phosphorylation lev- els of p38 mutants. The WT and active mutants of p38 were subjected to a Western blot using a specific antiphospho-p38 (Cell Signaling, catalog number 9211) before and after MKK6 treatment (lower image). Coomassie staining (upper image) indicates that equal amounts of each mutant or WT were loaded on the gel. It is apparent that all mutants can be phosphorylated by MKK6. All proteins exhibited some degree of phosphorylation prior to MKK6 treatment, including the WT p38.

Article Snippet: The WT and active mutants of p38 were subjected to a Western blot using a specific antiphospho-p38 (Cell Signaling, catalog number 9211) before and after MKK6 treatment (lower image).

Techniques: Western Blot, Phospho-proteomics, Staining, Mutagenesis

Journal: Journal of Biological Chemistry

Article Title: Active Mutants of the Human p38α Mitogen-activated Protein Kinase

doi: 10.1074/jbc.m404595200

Figure Lengend Snippet: FIG. 9. Stabilization model of the hydrophobic core in p38. Other than the hydrophobic core interactions, the two C- and L16- helices are mutually stabilized. The L16 loop at this region is mainly stabilized by the interactions formed among Tyr-69, Phe-327, and Trp- 337 (left). Substituting Tyr-69 is partially disrupting the L16 loop stability (middle). Conversely, by mutating Phe-327 the hydrophobic core cannot maintain the L16 conformational stability. Thus, the L16 segment, including the mutated residue, is highly flexible (right). The conformational flexibility at L16 is directly correlated with to the ac- tivity level, displayed in Fig. 2, attained by the various mutants.

Article Snippet: The WT and active mutants of p38 were subjected to a Western blot using a specific antiphospho-p38 (Cell Signaling, catalog number 9211) before and after MKK6 treatment (lower image).

Techniques: Residue

Journal: Journal of Biological Chemistry

Article Title: Active Mutants of the Human p38α Mitogen-activated Protein Kinase

doi: 10.1074/jbc.m404595200

Figure Lengend Snippet: FIG. 8. p38 structure. A, ribbon repre- sentation of p38 (Protein Data Bank code 1P38) using PyMol (49). Residues that were targeted by mutagenesis are labeled, colored, and represented as transparent space-filling spheres. The L16 loop/helix extension is shown in dark gray. Three residues identified by the HOG1 genetic screen (Tyr-69, Phe-327, and Trp-337) are in close structural prox- imity to form the hydrophobic core. The Asp-176 side chain is located on the phos- phorylation lip and extends to the solvent without any apparent interactions with the protein. B, tube representation of the hydrophobic core using MIDAS (50). In the unphosphorylated p38 (left), both Phe-327 and Trp-337 are contributed by the L16 segment (loop and helix), whereas Tyr-69 is donated by the C-helix. Phe-327 is part of a short 310 helical segment that is assumed to be stabilized by the hydro- phobic core, thus inducing low basal ac- tivity. Upon activation as seen in dually phosphorylated p38 (right), the short helical segment, including the phenylala- nine residue, is becoming disordered. The corresponding Tyr-72 and Trp-340 (cor- responding to Tyr-69 and Trp-337 in p38) maintain their structural positions because the C- and L16-helices are well stabilized by other intramolecular interactions.

Article Snippet: The WT and active mutants of p38 were subjected to a Western blot using a specific antiphospho-p38 (Cell Signaling, catalog number 9211) before and after MKK6 treatment (lower image).

Techniques: Mutagenesis, Labeling, Solvent, Activation Assay, Residue

Journal: Journal of Biological Chemistry

Article Title: Active Mutants of the Human p38α Mitogen-activated Protein Kinase

doi: 10.1074/jbc.m404595200

Figure Lengend Snippet: FIG. 10. Stereo view of the phosphorylated Thr-183 interactions in the activated p38. Only four of the six H-bond interactions formed by the phosphoryl group are shown for clarity. The H-bond formation with Arg-176 stabilizes a bulge in the phosphorylation lip. In the activated p38 form the phosphorylation lip goes through a substantial conformational change and is now stabilized in close proximity to the L16 region. It is also apparent that the side chain Asp-179 (corresponding to Asp-176 in p38) extends to the solvent and does not form any intramolecular interactions. The two aromatic residues, part of the hydrophobic core, are shown in blue. The L16 segment is shown in pink, and asterisks indicate the edges of the disordered segment. Phosphorylated Thr-183 (pThr 183) forms two hydrogen bonds with Arg-73 and one with Lys-69 to form the C-helix. Those two residues are adjacent to Tyr-72 (corresponding to Tyr-69 in p38), which is a part of the hydrophobic core. The image was rendered using PyMol (49).

Article Snippet: The WT and active mutants of p38 were subjected to a Western blot using a specific antiphospho-p38 (Cell Signaling, catalog number 9211) before and after MKK6 treatment (lower image).

Techniques: Phospho-proteomics, Solvent

Journal: Scientific Reports

Article Title: Up-regulation of ST18 in pemphigus vulgaris drives a self-amplifying p53-dependent pathomechanism resulting in decreased desmoglein 3 expression

doi: 10.1038/s41598-022-09951-x

Figure Lengend Snippet: Antibody-mediated DSG3 down-regulation affects p53 expression and activity in a p38MAPK-dependent manner. ( a ) NHEKs exposed to either AK23 or negative control antibody (NC) were stained for p53 (green) and DAPI (blue) (scale bar = 20um); ( b ) p53 protein expression in NHEKs exposed to either AK23 or negative control antibody (NC) was assessed using immunoblotting with anti-p53 antibody. β-actin served as a loading control (left panel). Protein levels were quantified and data was normalized to levels observed in negative control antibody-treated cells (right panel). Results represent the mean ± SE of four independent experiments (*p < 0.05 by 2-tailed t test); ( c ) NHEKs were transfected with a luciferase reporter construct under the regulation of a p53 binding site or with a control reporter, and were then treated either with AK23 antibody or negative control antibody (NC). Results represent the mean ± SE of three independent experiments (***p < 0.001 by 2-tailed t test); ( d ) NHEKs were transfected with a luciferase reporter construct under the regulation of a p53 binding site or a control reporter. Cells were additionally transfected with control (si-control) or p38MAPK -specific (si-p38) siRNAs. Twenty four hours post-transfection, cells were treated either with AK23 antibody (AK23 Ab) or negative control antibody (NC Ab). Results represent the mean ± SE of four independent experiments (*p < 0.05 by one way ANOVA test). Original blots are presented in Supplementary Fig. .

Article Snippet: Briefly, primary keratinocytes were co-transfected with the same pGL2 vectors, Renilla expression vector and control siRNA (Life Technologies, Carlsbad, CA, 452002) or a specific p38 alpha MAPK14-specific siRNA (Santa Cruz Biotechnology, Santa Cruz, CA, SC-29433).

Techniques: Expressing, Activity Assay, Negative Control, Staining, Western Blot, Control, Transfection, Luciferase, Construct, Binding Assay

Journal: Scientific Reports

Article Title: Up-regulation of ST18 in pemphigus vulgaris drives a self-amplifying p53-dependent pathomechanism resulting in decreased desmoglein 3 expression

doi: 10.1038/s41598-022-09951-x

Figure Lengend Snippet: ST18 drives a p53-dependent self-amplifying process promoting autoantibody-mediated membranal DSG3 down-regulation in pemphigus vulgaris. Genetically determined ST18 overexpression (1) enhances autoantibodies-induced DSG3 down-regulation in keratinocytes (2, orange arrow) which triggers p38MAPK-dependent (3) p53 activity (4), which in turn up-regulates ST18 promoter activity (5), thus setting the stage for a self-amplifying pathogenetic cycle in PV.

Article Snippet: Briefly, primary keratinocytes were co-transfected with the same pGL2 vectors, Renilla expression vector and control siRNA (Life Technologies, Carlsbad, CA, 452002) or a specific p38 alpha MAPK14-specific siRNA (Santa Cruz Biotechnology, Santa Cruz, CA, SC-29433).

Techniques: Over Expression, Activity Assay

Journal: Molecular Cancer

Article Title: IL-1β-induced activation of p38 promotes metastasis in gastric adenocarcinoma via upregulation of AP-1/c-fos, MMP2 and MMP9

doi: 10.1186/1476-4598-13-18

Figure Lengend Snippet: IL-1β promotes GA cell migration and invasion by activating p38. A : Western blots confirmed that p-p38 could be induced by 30 min stimulation with IL-1β in AGS and MKN-45 cell lines; the activation of p38 by IL-1β was inhibited by p38 inhibitor SB202190. B : Transfection of p38 siRNA knocked down p38 expression in both the two GA cell lines. C-F : Treatment of GA cells with IL-1β increased cell migration and invasion in vitro; these effects were inhibited by p38 siRNA or the p38 pathway inhibitor SB202190. ** P < 0.05 vs. scramble siRNA (Control siRNA)-transfected cells stimulated with IL-1β; △△ P < 0.05 vs. untransfected cells (wild type cells) stimulated with IL-1β. Bars indicate the mean ± SD number of cells per field of view (× 400 magnification) in the migration and invasion assays. G : Representative light microscope images of AGS and MKN-45 cell migration and invasion in the Transwell assays.

Article Snippet: To detect the expression of p-p38 in the 105 cases of GA tissues and in nude mice lung metastasic gatric cancer by immunohistochemistry (IHC), we used previously described methods [ - ], with the use of a specific anti-p-p38 antibody (#4631) (1:100 dilution, Cell Signaling Company, Danvers, MA, USA).

Techniques: Migration, Western Blot, Activation Assay, Transfection, Expressing, In Vitro, Control, Light Microscopy

Journal: Molecular Cancer

Article Title: IL-1β-induced activation of p38 promotes metastasis in gastric adenocarcinoma via upregulation of AP-1/c-fos, MMP2 and MMP9

doi: 10.1186/1476-4598-13-18

Figure Lengend Snippet: IL-1β upregulates MMP2 and MMP9 expression and activity by activating p38. A : RT-PCR analysis showed that MMP2 and MMP9 mRNA were upregulated in both AGS and MKN-45 cells in response to treatment with IL-1β; this effect was blocked by p38 siRNA or the p38 inhibitor SB202190. B : Quantification of the expression of MMP2 and MMP9 mRNA normalized to GAPDH mRNA. ** P < 0.05 vs. scramble siRNA-transfected cells stimulated with IL-1β. △△ P < 0.05 vs. untransfected cells stimulated with IL-1β. C: Gel zymography showed that MMP2 and MMP9 activity were upregulated in both AGS and MKN-45 cells in response to treatment with IL-1β; this effect was blocked by p38 siRNA or the p38 inhibitor SB202190. D: Immunocytochemical staining and confocal microscopy confirmed that IL-1β increased activation of p38 (red), and MMP2 and MMP9 (green) expression in MKN-45 GA cells; these effects were blocked by p38 siRNA or the p38 inhibitor SB202190.

Article Snippet: To detect the expression of p-p38 in the 105 cases of GA tissues and in nude mice lung metastasic gatric cancer by immunohistochemistry (IHC), we used previously described methods [ - ], with the use of a specific anti-p-p38 antibody (#4631) (1:100 dilution, Cell Signaling Company, Danvers, MA, USA).

Techniques: Expressing, Activity Assay, Reverse Transcription Polymerase Chain Reaction, Transfection, Zymography, Staining, Confocal Microscopy, Activation Assay

Journal: Molecular Cancer

Article Title: IL-1β-induced activation of p38 promotes metastasis in gastric adenocarcinoma via upregulation of AP-1/c-fos, MMP2 and MMP9

doi: 10.1186/1476-4598-13-18

Figure Lengend Snippet: IL-1β activates AP-1 activity through p38 pathway. A : Both GA cells were transfected with AP-1 reporter plasmid or AP-1 reporter plasmid together with scramble siRNA or p38 siRNA. AP-1 luciferase reporter gene activity was significantly increased by IL-1β stimulation in both AGS and MKN-45 cells; these effects were significantly inhibited by p38 siRNA in a dose-dependent manner and also inhibited by the p38 inhibitor SB202190. ** P < 0.05 vs. control siRNA (scramble siRNA) + AP-1 luc-transfected cells stimulated with IL-1β; ΔΔ P < 0.05 vs. AP-1 luc-transfected cells stimulated with IL-1β; relative luciferase activity was normalized to B-gal. B : IL-1β-induced p38-mediated activation of the MMP9 promoters is dependent on the AP-1-binding sites. ** P < 0.05 vs. transfected −670/MMP9 + control siRNA cells stimulated with IL-1β; ΔΔ P < 0.05 vs. transfected −670/MMP9 cells stimulated with IL-1β; ▼▼ P < 0.05 vs. transfected −570/MMP9 cells stimulated with IL-1β; relative luciferase activity was normalized to B-gal.

Article Snippet: To detect the expression of p-p38 in the 105 cases of GA tissues and in nude mice lung metastasic gatric cancer by immunohistochemistry (IHC), we used previously described methods [ - ], with the use of a specific anti-p-p38 antibody (#4631) (1:100 dilution, Cell Signaling Company, Danvers, MA, USA).

Techniques: Activity Assay, Transfection, Plasmid Preparation, Luciferase, Control, Activation Assay, Binding Assay

Journal: Molecular Cancer

Article Title: IL-1β-induced activation of p38 promotes metastasis in gastric adenocarcinoma via upregulation of AP-1/c-fos, MMP2 and MMP9

doi: 10.1186/1476-4598-13-18

Figure Lengend Snippet: Phosphorylated p38 is overexpressed in human GA, and the expression of p-p38 positively correlates with IL-1β, MMP2, MMP9 and c-fos expression in GA tissues. A : Overexpression of p-p38 was frequently observed in GA tissues, compared to the paired non-neoplastic gastric tissues. a , P-p38 was not detected or only weakly expressed in non-neoplastic gastric tissues. b to e : different intensities of positive p-p38 expression in GA tissues. B : Expression of p-p38 positively correlated with IL-1β, MMP2, MMP9, and c-fos expression in human GA tissues. GA tissue sample exhibiting stronger IL-1β expression and stronger p-p38, MMP2, MMP9 and c-fos expression; and weaker IL-1β expression and weak p-p38, MMP2, MMP9 and c-fos expression. C : The sum scores of positive staining intensity of IHC for p-p38. ** P < 0.05 vs. paired non- neoplastic gastric tissues.

Article Snippet: To detect the expression of p-p38 in the 105 cases of GA tissues and in nude mice lung metastasic gatric cancer by immunohistochemistry (IHC), we used previously described methods [ - ], with the use of a specific anti-p-p38 antibody (#4631) (1:100 dilution, Cell Signaling Company, Danvers, MA, USA).

Techniques: Expressing, Over Expression, Staining

Journal: Molecular Cancer

Article Title: IL-1β-induced activation of p38 promotes metastasis in gastric adenocarcinoma via upregulation of AP-1/c-fos, MMP2 and MMP9

doi: 10.1186/1476-4598-13-18

Figure Lengend Snippet: IL-1β stimulation increases the metastatic potential of GA cells in vivo via a p38–dependent mechanism. A : Representative lung metastases formed in different group mouse. B : Bars indicated the number of animals developed lung metastases. C : Representative results of HE staining of lung metastases. D : Bars indicated the mean ± SD number of metastatic foci in 6 of lung sections/per lung in mice that had developed lung metastases. △△ P < 0.05 vs. injected with scramble siRNA transfected cells plus IL-1β stimulation. E : RT-PCR analysis of p38, MMP2, MMP9 and c-fos mRNA expression in the lung metastases of different mice. F : IHC analysis of p-p38, MMP2, MMP9 and c-fos protein expression in the lung metastases; IL-1β induced elevated p-p38, MMP2, MMP9 and c-fos protein expression.

Article Snippet: To detect the expression of p-p38 in the 105 cases of GA tissues and in nude mice lung metastasic gatric cancer by immunohistochemistry (IHC), we used previously described methods [ - ], with the use of a specific anti-p-p38 antibody (#4631) (1:100 dilution, Cell Signaling Company, Danvers, MA, USA).

Techniques: In Vivo, Staining, Injection, Transfection, Reverse Transcription Polymerase Chain Reaction, Expressing, Paraffin-embedded Immunohistochemistry

Journal: Genes to cells : devoted to molecular & cellular mechanisms

Article Title: Mitogen-activated protein kinase p38 defines the common senescence-signalling pathway.

doi: 10.1046/j.1365-2443.2003.00620.x

Figure Lengend Snippet: Figure 1 p38 is activated in a telomere-independent manner in senescence. (A) Lysates obtained from young (at PD 40), senescent (at PD 60) WI-38 and young WI-38 cells treated with 30 µm anisomycin for 30 min were subjected to immunoblot analysis using anti-p38, anti-phosphorylated p38, anti-JNK and anti-phosphorylated JNK antibodies. Immunoprecipitated p38 was subjected to an immune- complex kinase assay using recombinant ATF-2 as the substrate. (B) Telomerase activity in hTERT-transfected MRC-5 cells at PD 60. The untransfected, parent MRC-5 cells at PD 40 and 60 serve as a negative control. The signal is sensitive to RNase pre-treatment of the extracts, indicating that it represents telomerase activity. The 65 bp product serves as the internal control to monitor PCR efficiency. (C) Telomere lengths of hTERT-transfected MRC-5 cells at PD 60 and the control MRC-5 cells at PD 40 and 60. Genomic DNA was digested with HinfI and the blotted filter was hybridized with radiolabelled (TTAGGG)n. (D) Immunoblot analysis of p38 MAP kinase (p38, total p38; and p38-P, phosphorylated p38) in untransfected MRC-5 cells at PD 40 and 60, and hTERT-transfected MRC-5 cells at PD 60.

Article Snippet: Primers for stromelysin cDNA amplification were 5′-ATATCTAGAAAACTACTACGACCTC-3′ and 5′-TCAAAAGGGTAAAAGTCTCCATGTT-3′. p38 MAP kinase assays Immunoprecipitation and kinase reaction were performed as previously described (Lin et al. 1998) with a specific antibody against p38 using recombinant ATF-2 protein (Santa Cruz).

Techniques: Western Blot, Immunoprecipitation, Immune Complex Kinase Assay, Recombinant, Activity Assay, Transfection, Negative Control, Control

Journal: Genes to cells : devoted to molecular & cellular mechanisms

Article Title: Mitogen-activated protein kinase p38 defines the common senescence-signalling pathway.

doi: 10.1046/j.1365-2443.2003.00620.x

Figure Lengend Snippet: Figure 2 Effects of p38 inactivation on telomere-dependent senescence. (A) Representative growth curves documenting the effects of SB203580 on replicative growth arrest. Young and senescent cells were fed daily fresh medium containing 10 µm SB203580 (SB) or DMSO alone. Note that the y-axis is shown in the logarithmic scale. (B) Effects of SB203580 on BrdU incorporation in young and senescent WI-38 cells. Cells were precultured with 10 µm SB203580 (+SB), or solvent alone (–SB) for 24 h, and then labelled with BrdU for 72 h. SB203580 or solvent was fed daily during the labelling period. BrdU uptake (% BrdU-positive cells) was scored. The averages and standard deviations of data from at least three separate experiments are shown. *P < 0.05. (C) SA-β-gal staining of SB203580-treated and mock-treated senescent cells. Senescent cells were fed daily 10 µm SB203580 or solvent alone for 6 days, and then fixed and stained for SA-β-gal activity. Percentages of cells positive for SA-β-gal activity after six days of SB203580 or mock treatment are indicated. (D) Proliferation assay of mock and MKK6AA WI-38 cells. The time point when the drug selection was completed was designated as PD 0.

Article Snippet: Primers for stromelysin cDNA amplification were 5′-ATATCTAGAAAACTACTACGACCTC-3′ and 5′-TCAAAAGGGTAAAAGTCTCCATGTT-3′. p38 MAP kinase assays Immunoprecipitation and kinase reaction were performed as previously described (Lin et al. 1998) with a specific antibody against p38 using recombinant ATF-2 protein (Santa Cruz).

Techniques: BrdU Incorporation Assay, Solvent, Staining, Activity Assay, Proliferation Assay, Selection

Journal: Genes to cells : devoted to molecular & cellular mechanisms

Article Title: Mitogen-activated protein kinase p38 defines the common senescence-signalling pathway.

doi: 10.1046/j.1365-2443.2003.00620.x

Figure Lengend Snippet: Figure 3 Effects of p38 inactivation on telomere-independent senescence in MEF cells. Proliferation assay for SB203580-treated (+SB) and mock-treated (NT) MEFs that were passaged according to the 3T3 protocol. An aliquot of SB203580-treated cells was removed and cultured in the absence of SB203580 at PD 20 (–SB-1) or PD 35 (–SB- 2). Immunoblot analysis of p38 MAP kinase in early passage (PD 3), and late-passage (PD 13) MEFs.

Article Snippet: Primers for stromelysin cDNA amplification were 5′-ATATCTAGAAAACTACTACGACCTC-3′ and 5′-TCAAAAGGGTAAAAGTCTCCATGTT-3′. p38 MAP kinase assays Immunoprecipitation and kinase reaction were performed as previously described (Lin et al. 1998) with a specific antibody against p38 using recombinant ATF-2 protein (Santa Cruz).

Techniques: Proliferation Assay, Cell Culture, Western Blot

Journal: Genes to cells : devoted to molecular & cellular mechanisms

Article Title: Mitogen-activated protein kinase p38 defines the common senescence-signalling pathway.

doi: 10.1046/j.1365-2443.2003.00620.x

Figure Lengend Snippet: Figure 4 p38 is activated even after withdrawal of H2O2 treatment. (A) Representative growth curves documenting the effects of SB203580 on H2O2-induced growth arrest. Treated WI-38 cells were fed daily a medium containing 10 µm SB203580 (SB) or an equivalent volume of DMSO. Note that the y-axis is shown in the logarithmic scale. (B) Percentage of cells positive for SA-β-gal activity after six days of SB203580 (+) or solvent (–) treatment. (C) Immunoblot analysis of p38 MAP kinase (p38, total p38; and p38-P, phosphorylated p38) in WI-38 cells on Day 8 after 2 h H2O2 treatment (H2O2) or no treatment (NT).

Article Snippet: Primers for stromelysin cDNA amplification were 5′-ATATCTAGAAAACTACTACGACCTC-3′ and 5′-TCAAAAGGGTAAAAGTCTCCATGTT-3′. p38 MAP kinase assays Immunoprecipitation and kinase reaction were performed as previously described (Lin et al. 1998) with a specific antibody against p38 using recombinant ATF-2 protein (Santa Cruz).

Techniques: Activity Assay, Solvent, Western Blot

Journal: Genes to cells : devoted to molecular & cellular mechanisms

Article Title: Mitogen-activated protein kinase p38 defines the common senescence-signalling pathway.

doi: 10.1046/j.1365-2443.2003.00620.x

Figure Lengend Snippet: Figure 5 p38 is activated even after withdrawal of tamoxifen- induced Raf activation. (A) Representative growth curves docu- menting the effects of PD98059 and SB203580 on Raf-induced growth arrest. ∆Raf-1:ER[DD]-expressing WI-38 cells were treated with either 10 nm 4-hydroxy-tamoxifen (4-HT) or an equivalent volume of ethanol (EtOH) for 4 days in the presence of 25 µm PD98059, 25 µm SB203580 or an equivalent volume of DMSO. (B) Percentage of cells positive for SA-β-gal activity after four days of SB203580, PD98059, or mock (DMSO) treatment. Both 4- HT-treated (4-HT) and solvent-treated (EtOH) cells were examined. (C) Kinetics of p38 activation after induction of active Raf kinase with 10 nm 4-HT in ∆Raf-1:ER[DD]-expressing WI-38 cells. Cell lysates were prepared at indicated time points, and subjected to immunoblot analysis using specific antibodies. (D) Kinetics of p38 activation after induction of active Raf kinase with 1 µm 4-HT in ∆Raf-1:ER[DD]-expressing WI-38 cells. Cells were treated with (4-HT(+)) or without (4-HT(–)) 1 µm 4-HT for 24 h. Then, the 4-HT-containing culture medium was replaced with a 4-HT(–) fresh medium, and cultured for another 72 h (4-HT(–)).

Article Snippet: Primers for stromelysin cDNA amplification were 5′-ATATCTAGAAAACTACTACGACCTC-3′ and 5′-TCAAAAGGGTAAAAGTCTCCATGTT-3′. p38 MAP kinase assays Immunoprecipitation and kinase reaction were performed as previously described (Lin et al. 1998) with a specific antibody against p38 using recombinant ATF-2 protein (Santa Cruz).

Techniques: Activation Assay, Expressing, Activity Assay, Solvent, Western Blot, Cell Culture

Journal: Genes to cells : devoted to molecular & cellular mechanisms

Article Title: Mitogen-activated protein kinase p38 defines the common senescence-signalling pathway.

doi: 10.1046/j.1365-2443.2003.00620.x

Figure Lengend Snippet: Figure 7 Effects of papilloma virus E6 and E7 on senescence phenotypes induced by p38 activation. (A) Cell-cycle analysis of control, E6, E7 and E6/E7 cells that were transduced with mock retrovirus (mock) or MKK6EE-expressing retrovirus (MKK6EE). Cells on Day 6 after drug selection were labelled with BrdU for 4 h. Cells were fractionated by BrdU labelling and PI staining. (B) Percentage of SA-β-gal-positive MKK6EE-expressing cells.

Article Snippet: Primers for stromelysin cDNA amplification were 5′-ATATCTAGAAAACTACTACGACCTC-3′ and 5′-TCAAAAGGGTAAAAGTCTCCATGTT-3′. p38 MAP kinase assays Immunoprecipitation and kinase reaction were performed as previously described (Lin et al. 1998) with a specific antibody against p38 using recombinant ATF-2 protein (Santa Cruz).

Techniques: Virus, Activation Assay, Cell Cycle Assay, Control, Transduction, Expressing, Selection, Staining

Journal: Journal of Cancer Research and Clinical Oncology

Article Title: Effects of VEGFR1 + hematopoietic progenitor cells on pre-metastatic niche formation and in vivo metastasis of breast cancer cells

doi: 10.1007/s00432-018-2802-6

Figure Lengend Snippet: HPCs enhances the invasive growth of MDA-MB-435s-HM cells. HPCs were co-cultured with MDA-MB-435s-HM cells, and cells were treated with an inhibitor of LY-294002, PD98059, SB431542, CP690550, or BIRB796. The invasive growth was assessed based on a Micro PET photographs, as area of nodules in liver formed by MDA-MB-435s-HM cells or Masson staining indicating the invasive growth of cells. Quantitative results from b Micro PET photographs; c fold difference in radioactivity between liver and blood; d inhibitory rates of inhibitors on 18 F-FDG intensity or e invasive growth of cells. Definitions: inhibitor of MMP-9, tissue inhibitor of metalloproteinase-1 (TIMP-1) shown to be a natural inhibitor of MMP-9; anti-CD31 antibody, anti-human CD31 antibody recognizing the D2 extracellular portion of CD31 to block its function; inhibitor for PDGFR-α, PDGFR tyrosine kinase inhibitor III shown to be an ATP-competitive inhibitor of PDGFR-α. * P < 0.05. White arrow in Figure indicates the nodules in mouse liver

Article Snippet: Small molecular inhibitors, LY294002 (a specific inhibitor of PI3K/AKT pathway, cat #S1105), PD98059 (a specific inhibitor of MEK/ERK pathway, cat #S1177), CP690550 (a specific inhibitor of Jak/STAT pathway, cat #S2789), SB431542 (a specific inhibitor of TGFβ/Smad pathway, cat #S1067), and BIRB796 (a specific inhibitor of p38 MAPK pathway, cat #S1574) were purchased from Selleck Corporation (Houston, TX, USA).

Techniques: Cell Culture, Micro-PET, Staining, Radioactivity, Blocking Assay