Journal: Oncogene

Article Title: The fungicide ciclopirox inhibits lymphatic endothelial cell tube formation by suppressing VEGFR-3-mediated ERK signaling pathway.

doi: 10.1038/onc.2010.590

Figure Lengend Snippet: Figure 5 CPX inhibits VEGFR-3-mediated ERK1/2 pathway. (a, b) CPX inhibited phosphorylation of ERK1/2, but not Akt, JNK and p38 mitogen-activated protein kinase, in LECs in a concentration- and time-dependent manner. LECs treated with CPX (0–5 mM) for 24 h (a) or CPX (5 mM) for 0–24 h (b) were harvested and subjected to western blot analysis with indicated antibodies. b-Tubulin was used as a loading control. (c) Overexpression of VEGFR-3 conferred resistance to CPX inhibition of ERK1/2 phosphorylation in LECs. LEC/V (control) and LEC/VEGFR-3 were treated with or without CPX (5 mM) for 24 h, followed by western blotting with indicated antibodies. (d) Downregulation of VEGFR-3 mimicked the effect of CPX, inhibiting phosphorylation of ERK1/2 in LECs. LECs, infected with lentiviral shRNAs to VEGFR-3 and GFP (control), respectively, were treated with CPX (5 mM) for 24 h, followed by western blotting with indicated antibodies.

Article Snippet: The primary antibodies used included antibodies to VEGFR-3, glyceraldehyde 3-phosphate dehydrogenase, Akt, ERK2, JNK1, phospho-JNK (Thr183/ Tyr185), p38, phospho-p38 (Thr180/Tyr182), MKK1, Flag (Santa Cruz Biotechnology, Santa Cruz, CA, USA), phosphoERK1/2 (Thr202/Tyr204), phospho-Akt (Ser473) (Cell Signaling, Beverly, MA, USA) and b-tubulin (Sigma).

Techniques: Phospho-proteomics, Concentration Assay, Western Blot, Control, Over Expression, Inhibition, Infection

Journal: Molecular medicine reports

Article Title: Endothelin-1 downregulates Mas receptor expression in human cardiomyocytes.

doi: 10.3892/mmr.2013.1577

Figure Lengend Snippet: Figure 2. Effects of endothelin-1 (ET-1) on human Mas promoter activities and on Mas mRNA level in human cardiomyocytes treated with siRNA against p38 mitogen-activated protein kinase (MAPK). (A) Human cardiomyocytes were transfected with human Mas promoter-luciferase reporter plasmids and then treated with ET-1 (10 or 30 nM) for 4.5 h. Twenty‑four hours later, luciferase assays were performed. Luciferase activities were expressed as fold changes to that of untreated control cells (designated as 1). (B) Human cardiomyocytes with or without transfection of p38 MAPK siRNA were treated with ET-1 (30 nM) for 4.5 h. Mas mRNA level of treated cells was shown as fold changes to that of untreated control cells (designated as 1). aP<0.05 compared with untreated control cells and bP<0.05 compared with 10 nm ET-1 treatment.

Article Snippet: Furthermore, anti-phospho-p38 (Thr180/Tyr182) (#9212) and anti-p38 (#8690) antibodies were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA).

Techniques: Transfection, Luciferase, Control

Journal: Molecular medicine reports

Article Title: Endothelin-1 downregulates Mas receptor expression in human cardiomyocytes.

doi: 10.3892/mmr.2013.1577

Figure Lengend Snippet: Figure 5. Western blot analysis of phosphorylated p38 mitogen-activated protein kinase (MAPK) levels in human cardiomyocytes. (A) Human cardiomyocytes were treated with ET-1 (1, 5, 10, 20 and 30 nM) with or without BQ123 (1 µM), BQ788 (1 µM), PD169316 (25 µM) or p38 MAPK siRNA for 4.5 h. Lysates from untreated human cardiomyocytes were used as a control (lane 1). Concentrations of ET‑1 were as follows: Lane 2, 1 nM; lane 3, 5 nM; lane 4, 10 nM; lane 5, 20 nM; lane 6, 30 nM; lane 7, 30 nM and BQ123 (1 µM); lane 8, 30 nM and BQ788 (1 µM); lane 9, 30 nM and PD169316 (25 µM); lane 10, 30 nM and p38 siRNA. β-actin blotting was used as a loading control. (B) Phosphorylated p38 (Thr180/Tyr182) and total p38 MAPK levels were measured by densitometry. The density of the phosphorylated p38 (Thr180/Tyr182) (pp38) MAPK blot was normalized against that of total p38 MAPK levels to obtain a relative density, which was expressed as fold changes to that of untreated control cells (designated as 1). aP<0.05 compared with untreated control cells; bP<0.05 compared with 5 nM ET-1 treatment and cP<0.05 compared with 10 nM ET-1 treatment.

Article Snippet: Furthermore, anti-phospho-p38 (Thr180/Tyr182) (#9212) and anti-p38 (#8690) antibodies were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA).

Techniques: Western Blot, Control

Journal: Molecular medicine reports

Article Title: Endothelin-1 downregulates Mas receptor expression in human cardiomyocytes.

doi: 10.3892/mmr.2013.1577

Figure Lengend Snippet: Figure 4. Saturation binding assay of Mas receptor on the cell membrane of human cardiomyocytes. Human cardiomyocytes were treated with ET-1 (1, 5, 10, 20 and 30 nM) with or without BQ123 (1 µM), BQ788 (1 µM), PD169316 (25 µM) or p38 mitogen‑activated protein kinase siRNA for 4.5 h. Twenty‑four h later, saturation binding assays were conducted using increasing concentrations of 125I-Ang-(1-7) (1-22 nM) on cell membranes. A single-site receptor binding model provided the best fit for data analysis. Disintegrations per min (dpm) data were normalized against the cell number (per 20000 cells) and shown as a percentage of that of untreated control cells (designated as 100%). aP<0.05 compared with untreated control cells; bP<0.05 compared with 5 nM ET-1 treatment and cP<0.05 compared with 10 nM ET-1 treatment.

Article Snippet: Furthermore, anti-phospho-p38 (Thr180/Tyr182) (#9212) and anti-p38 (#8690) antibodies were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA).

Techniques: Saturation Assay, Membrane, Binding Assay, Control

Journal: Biochimica et biophysica acta

Article Title: Mechanical regulation of cancer cell apoptosis and autophagy: roles of bone morphogenetic protein receptor, Smad1/5, and p38 MAPK.

doi: 10.1016/j.bbamcr.2013.08.023

Figure Lengend Snippet: Fig. 5. Shear-induced autophagy and apoptosis in Hep3B cells are mediated by p38 MAPK. (A) Hep3B cells were maintained under static conditions (C) or subjected to LSS at 0.5 dyn/cm2 for the indicated times, and the levels of phospho-ERK (p-ERK), phospho-JNK (p-JNK), and phospho-p38 MAPK (p-p38) were examined. (B and C) Hep3B cells were transfected with the designated siRNAs for 48 h and then maintained as controls or subjected to LSS at 0.5 dyn/cm2 for 1 h. (D) Hep3B cells were transfected with control (siCL) or p38 MAPK-specific siRNA (sip38) for 48 h. A sample of siCL-transfected cells was pre-treated with SB203580 for 1 h. All cells were maintained under static conditions or subjected to LSS for 24 h. The expressions of the cleaved caspase-9 (c-Casp9), caspase-3 (c-Casp3), PARP (c-PARP), and LC3B proteins were examined. (E) Hep3B cells were transfected with the designated siRNAs for 48 h and then maintained as controls or subjected to LSS at 0.5 dyn/cm2 for 24 h. The percentage of cell death was calculated. The results are representative of three independent experiments with similar results. *, P b 0.05 sheared cells vs. static cells; #, P b 0.05 cells transfected with BMPRIB-, Smad1/5-, or p38 MAPK-specific siRNA vs. control siRNA.

Article Snippet: Control, Atg5, Smad1, Smad5, BMPRIA, BMPRIB, and BMPRII-specific small interfering RNAs (siRNA) were purchased from Invitrogen Technology. p38 MAPK-specific siRNA was purchased from Cell Signaling Technology.

Techniques: Shear, Transfection, Control

Journal: Nucleus (Austin, Tex.)

Article Title: Depletion of lamina-associated polypeptide 1 from cardiomyocytes causes cardiac dysfunction in mice.

doi: 10.4161/nucl.29227

Figure Lengend Snippet: Figure 6. Mitogen-activated protein kinase signaling in hearts of H-CKO mice with cardiomyocyte-selective depletion of LAP1. (A) Immunoblots using antibodies against phosphorylated ERK1/2 (p-ERK1/2), total ERK1/2 (t-ERK1/2), phosphorylated JNK (p-JNK), total JNK (t-JNK), phosphorylated p38alpha (p-p38), and total p38alpha (t-p38) and GAPDH of protein extracts from hearts of control and H-CKO mice at 20 weeks of age. Each lane contains an extract from a different mouse. (B) Bar graphs showing quantification of the ratio of the phosphorylated protein signals to their respective total protein signals. Values are means ± standard errors (control n = 3; H-CKO n = 3). *P < 0.05, n.s. = not significant. Graphs are presented as fold change over controls. (C) Means ± standard errors of relative expression of mRNAs encoded by Atf2, Atf4, Nfatc4, and Elk4, genes activated by phosphorylated mitogen-activated protein kinases, in hearts of control (n = 4) and H-CKO (n = 4) mice at 20 weeks of age. **P < 0.01, ***P < 0.001.

Article Snippet: The primary antibodies used for immunoblotting were against LAP1,21 GAPDH (Ambion #AM4300), total ERK1/2 (Santa Cruz Biotechnologies Inc #SC-94), phosphorylated ERK1/2 (Cell Signaling #9101), total JNK (Santa Cruz Biotechnologies Inc #SC-474), phosphorylated JNK (Cell Signaling, #9251), total p38α (Cell Signaling, #9212), phosphorylated p38α (Cell Signaling, #4511).

Techniques: Western Blot, Control, Expressing

Journal: Leukemia

Article Title: Notch3-mediated regulation of MKP-1 levels promotes survival of T acute lymphoblastic leukemia cells.

doi: 10.1038/leu.2010.323

Figure Lengend Snippet: Figure 1 Higher ERK1/2 and p38 phosphorylation levels in dormant compared with aggressive T-ALL tumor samples inversely correlate with MKP-1 expression levels. (a) Detection of total and phosphorylated levels of ERK1/2, p38 and MKP-1 by western blot analysis of lysates from dormant or aggressive tumor samples formed by s.c. injection of MOLT-3 cells in NOD/SCID mice in the absence or the presence of exogenous bFGF (100 ng/ml), respectively.21 Membranes were probed with anti-a-tubulin as a loading control. Five representative samples per group are shown. (b) Columns report the mean values±s.d. of phosphorylated ERK (P-ERK)/ERK, P-p38/p38 (left), MKP-1/a-tubulin (right) ratios in all samples analyzed (n ¼ 10 per group). Samples were normalized to the ratio measured in the weakest one of the series, which was set at 1. Signal intensity was measured using a Bio-Rad XRS chemioluminescence detection system. *Po0.05 compared with dormant tumor values. (c) Expression of MKP-1 mRNA is similar in growing compared with dormant tumors. Total RNA was extracted from growing and dormant tumors (n ¼ 7 samples per group) and MKP-1 expression was determined by quantitative PCR. Columns represent mean values±s.d. MOLT-3 cells in vitro were used as reference sample to calculate the relative expression indicated in the panels. (d) MKP-1 is broadly expressed in T-ALL cell lines. Western blot analysis of MKP-1 in a panel of T-ALL cell lines; a-tubulin was used as loading control.

Article Snippet: P-p38 levels were analyzed by incubating 1 106 permeabilized cells with rabbit anti-P-p38 primary mAb (Cell Signaling), followed by labelling with goat anti-rabbit Alexa Fluor 488 secondary Ab (Invitrogen).

Techniques: Phospho-proteomics, Expressing, Western Blot, Injection, Control, Real-time Polymerase Chain Reaction, In Vitro

Journal: Leukemia

Article Title: Notch3-mediated regulation of MKP-1 levels promotes survival of T acute lymphoblastic leukemia cells.

doi: 10.1038/leu.2010.323

Figure Lengend Snippet: Figure 2 Notch3 signalling regulates MKP-1 expression. (a) Treatment of T-ALL cell lines with GSIs lowers MKP-1 protein expression. MOLT-3, Jurkat, DND41 and CEM cells were treated for 72 h with CompE (10 mM) or solvent (dimethylsulphoxide (DMSO)) before western blot analysis. Columns represent mean values±s.d. of n ¼ 3 experiments. Cells cultivated in vitro in the presence of DMSO were used as reference sample to calculate the relative expression indicated in the panels. (b) Forced Notch3 ICD expression affects MKP-1 protein expression in MOLT-3 cells. Western blot analysis of MOLT-3 cells transfected by a Notch3 ICD expression plasmid (N3-ICD) versus control cells (enhanced green fluorescent protein (EGFP)). Density values were normalized to the ratio measured in the control sample, which was set at 1. (c) Notch3 silencing reduces MKP-1 expression and activates p38 in T-ALL cell lines. Left panel, western blot analysis of MOLT-3 cells transduced by lentiviral vectors encoding two different Notch3-specific shRNA (shN3/1 and shN3/2) or the control shRNA. In all samples analyzed, density values were normalized to the ratio measured in the control sample, which was set at 1. Mid panel: following shN3/1 transduction of DND41 cells, Notch3, MKP-1, p38, P-p38 and a-tubulin levels were analyzed by immunoblotting, and quantification was performed by P-p38 AlphaScreen SureFire kit (right panel), as described in section ‘Materials and methods’. Results are expressed as mean±s.d. of two independent experiments; *Po0.05.

Article Snippet: P-p38 levels were analyzed by incubating 1 106 permeabilized cells with rabbit anti-P-p38 primary mAb (Cell Signaling), followed by labelling with goat anti-rabbit Alexa Fluor 488 secondary Ab (Invitrogen).

Techniques: Expressing, Solvent, Western Blot, In Vitro, Transfection, Plasmid Preparation, Control, shRNA, Transduction, Amplified Luminescent Proximity Homogenous Assay

Journal: Leukemia

Article Title: Notch3-mediated regulation of MKP-1 levels promotes survival of T acute lymphoblastic leukemia cells.

doi: 10.1038/leu.2010.323

Figure Lengend Snippet: Figure 4 MKP-1 modulates apoptosis of T-ALL cell lines. (a) Left panel: MKP-1 is expressed at higher levels in MOLT-3 primary cultures established from growing (GT) compared with dormant (DT) tumors by Western blot analysis. Right panel: Reduced apoptosis in primary cultures from GT compared with DT tumors following 6-h treatment with anysomicin (0.67 mg/ml). Apoptosis was evaluated by Annexin V staining and flow cytofluorimetric analysis. The columns report the mean values±s.d. of three independent experiments. *Po0.05 compared with untreated controls. (b) Transduction of MOLT-3 cells with lentiviral vectors encoding human MKP-1 (cytomegalovirus (CMV)–MKP-1) or shRNA targeting MKP-1 (shMKP-1) modulates MKP-1 expression levels in MOLT-3 cells, compared with controls (CMV; shRNA). The panels show the results of immunofluorescence analysis of MKP-1 expression in MOLT-3 cells transduced by the indicated constructs. Magnification, 200. (c) Detection of total and phosphorylated levels of JNK1-2 and p38 by Western blot analysis of lysates from MOLT-3 cells bearing modulated MKP-1 levels, treated or not with anisomycin. Membranes were probed with anti-b-actin as a loading control. One representative experiment of three performed is shown. (d) Measurements of apoptosis by Annexin V staining of T-ALL cells bearing increased (left panel) or reduced MKP-1 levels (right panels). MOLT-3 cells were treated for 6 h with anysomicin, 24 h with cisplatin (6 mg/ml), or 48-h serum starvation (fetal calf serum (FCS)) before staining with Annexin V–fluorescein isothiocyanate (FITC) and cytofluorimetric analysis. The columns report the mean values±s.d. of four independent experiments. *Po0.05 compared with controls. (e) Measurements of apoptosis of T-ALL cells bearing reduced MKP-1 levels following treatment with chemotherapeutics. Cell lines were treated for 24 h (MOLT-3) or 48 h (DND41) with cytarabine (20 nM), vincristine (10 mg/ml) or methotrexate (MTX, 300 nM) before staining with Annexin V–FITC and cytofluorimetric analysis. The columns report the mean values±s.d. of four independent experiments. *Po0.05 compared with controls.

Article Snippet: P-p38 levels were analyzed by incubating 1 106 permeabilized cells with rabbit anti-P-p38 primary mAb (Cell Signaling), followed by labelling with goat anti-rabbit Alexa Fluor 488 secondary Ab (Invitrogen).

Techniques: Western Blot, Staining, Transduction, shRNA, Expressing, Construct, Control

Journal: Leukemia

Article Title: Notch3-mediated regulation of MKP-1 levels promotes survival of T acute lymphoblastic leukemia cells.

doi: 10.1038/leu.2010.323

Figure Lengend Snippet: Figure 5 MKP-1 attenuation modulates apoptosis and P-p38 levels in vivo. MOLT-3 or DND41 cells bearing normal or reduced MKP-1 levels were mixed with Matrigel and bFGF and injected s.c. into NOD/SCID mice (six animals per group). Before injection, cells were subjected to measurements of MKP-1 RNA levels, which indicated reduction of MKP-1 expression by 50% in shRNA targeting MKP-1 (shMKP-1)-transduced cells compared with shRNA controls (not shown). (a) Macroscopical analysis of s.c. MOLT-3 and DND41 tumors, 3-week after injection. (b) Measurements of apoptosis by Annexin V staining of MOLT-3 and DND41 tumors bearing normal or reduced MKP-1 levels. (c) Cytofluorimetric analysis of P-p38 levels in DND41 xenografts. The columns report the mean values±s.d. of 5–8 samples per group. *Po0.05 compared with controls.

Article Snippet: P-p38 levels were analyzed by incubating 1 106 permeabilized cells with rabbit anti-P-p38 primary mAb (Cell Signaling), followed by labelling with goat anti-rabbit Alexa Fluor 488 secondary Ab (Invitrogen).

Techniques: In Vivo, Injection, Expressing, shRNA, Staining

Journal: Leukemia

Article Title: Notch3-mediated regulation of MKP-1 levels promotes survival of T acute lymphoblastic leukemia cells.

doi: 10.1038/leu.2010.323

Figure Lengend Snippet: Figure 7 Expression of MKP-1 in primary T-ALL cells and correlation with the other molecular variables. (a) p38 phosphorylation in pediatric T-ALL samples inversely correlates with MKP-1 expression levels by western blot analysis. Membranes were probed with anti-b-actin as a loading control. (b) Notch 3 transcript levels by quantitative PCR in T-ALL samples characterized by low versus high MKP-1 levels. Results were normalized to the Notch3 transcript level in samples PDTALL6, which was set at 1. (c) Treatment of T-ALL cells with a g-secretase inhibitor (CompE) lowers MKP-1 protein expression. Primary cells were treated for 72 h with CompE (10 mM) or solvent (dimethylsulphoxide (DMSO)) before western blot analysis. Cells cultivated in vitro in the presence of DMSO were used as reference sample. Columns represent mean values±s.d. of the pooled experiments. *Po0.05.

Article Snippet: P-p38 levels were analyzed by incubating 1 106 permeabilized cells with rabbit anti-P-p38 primary mAb (Cell Signaling), followed by labelling with goat anti-rabbit Alexa Fluor 488 secondary Ab (Invitrogen).

Techniques: Expressing, Phospho-proteomics, Western Blot, Control, Real-time Polymerase Chain Reaction, Solvent, In Vitro