Journal: BMC Cancer

Article Title: IL-1β induces IL-6 production and increases invasiveness and estrogen-independent growth in a TG2-dependent manner in human breast cancer cells

doi: 10.1186/s12885-016-2746-7

Figure Lengend Snippet: IL-1β induced IL-6 production in MCF7_TG2 cells through the IRAK1, NF-kB, JNK, and PI3K signaling pathway. a MCF7_TG2 cells were treated with IL-1β (10 ng/ml) in the presence of IRAK1/4 inhibitor (20 μM), a NF-kB inhibitor (Bay11-7082, 10 μM), a JNK inhibitor (SP600125, 10 μM), an ERK inhibitor (PD98059, 10 μM), a p38 MAPK inhibitor (SB209580, 10 μM), or a PI3K inhibitor (LY294002, 10 μM) for 48 h. IL-6 levels in culture supernatants were measured by ELISA. b MCF7_Cont and MCF7_TG2 cells were treated with IL-1β (10 ng/ml) for the indicated times. Phospho-p65, p65, Ik-Bα, phospho-JNK, and JNK were detected by Western blot. c MCF7_Cont and MCF7_TG2 cells were treated with IL-1β (10 ng/ml) for the indicated times. IRAK1, IRAK2, and TRAF6 were detected by Western blot. d MCF7_Cont and MCF7_TG2 cells were co-transfected with p3kB-Luc and pRL-TK reporter constructs for 24 h then treated with IL-1β (10 ng/nl) for 18 h. All data shown are representative of three independent experiments

Article Snippet: The following signaling inhibitors were added to some cultures 1 h before IL-1β treatment; IRAK1/4 inhibitor (20 μM; Calbiochem), NF-kB inhibitor (Bay-117082, 10 μM; Calbiochem), JNK inhibitor (SP600125, 10 μM; Calbiochem), ERK inhibitor (PD98059, 10 μM; Alomone labs, Jerusalem, Israel), p38 MAPK inhibitor (SB209580, 10 μM; Cell signaling), and PI3K inhibitor (LY294002, 10 μM; Alomone labs).

Techniques: Enzyme-linked Immunosorbent Assay, Western Blot, Transfection, Construct

Journal: Cancers

Article Title: TRPC3 Regulates the Proliferation and Apoptosis Resistance of Triple Negative Breast Cancer Cells through the TRPC3/RASA4/MAPK Pathway

doi: 10.3390/cancers11040558

Figure Lengend Snippet: TRPC3 blockade induced apoptosis in MDA-MB-231 cells through activation of ERK 1/2. ( A ) decrease in the percentage of cell proliferation in response to Pyr3 (1.0 μM for 72 h) was attenuated by pre-treatment with ERK1/2 inhibitor PD98059 (5.0 μM for 24 h) as measured by MTT assay. Pre-treatment of MDA-MB-231 cells with p38 MAPK inhibitor SB202190 (1.0 μM for 24 h) and JNK inhibitor SP600125 (1.0 μM for 24 h) did not reverse the effect of Pyr3. Values are mean ± SEM ( n = 3). ** p < 0.01 and *** p < 0.001; ( B ) cell density and cell morphology of the four treatment groups (DMSO only, DMSO followed by Pyr3, PD98059 followed by Pyr3 and PD98059 only) were observed under phase-contrast microscope. Scale bar: 100 μm; ( C ) representative Western blots showing that increased level of cleaved PARP and phosphorylated ERK1/2 proteins induced by Pyr3 was attenuated by pre-treatment with ERK1/2 inhibitor PD98059 (5.0 μM for 24 h).

Article Snippet: SP600125 (JNK inhibitor, 1 μmol/L, Tocris), PD98059 (MEK-ERK inhibitor, 5 μmol/L, Tocris), and SB202190 (p38 MAPK inhibitor, 1 μmol/L, Tocris) were used to treat cells for 24 h prior to Pyr3 exposure.

Techniques: Activation Assay, MTT Assay, Microscopy, Western Blot

Journal: Oncogene

Article Title: Stat3-dependent induction of BATF in M1 mouse myeloid leukemia cells.

doi: 10.1038/sj.onc.1205918

Figure Lengend Snippet: Figure 3 (a) M1 cells were treated with 50 mM PD98059 and sti- mulated with LIF for 1 h. Cell lysates were immunoblotted with anti-phospho-MAPK antibody (apMAPK) (Cell Signaling) or anti-MAPK antibody (aMAPK) (Santa Cruz Biotechnology). (b) M1 cells were stimulated with LIF plus 50 mM PD98059 or (c) LIF plus 10 ng/ml cyclohexamide (CHX) and induction of BATF mRNA was examined by RNA blot hybridization

Article Snippet: Interestingly, in parallel with the expression pattern of BATF, which peaks between 3 and 6 h after LIF exposure (Figure Figure 2 (a) M1 cells, M1/705F cells and M1/JAB cells were stimulated with LIF for 1 h and cell lysates were immunoblotted with anti-phospho-Y705 Stat3 antibody (ap705YStat3) (New England Biolabs) or anti-Stat3 antibody (aStat3) (New England BioLabs). (b) M1 cells, M1/705F cells and M1/JAB cells were stimulated with LIF for 1 h and induction of BATF mRNA was analysed by RNA blot hybridization. (c) M1/Stat3ER cells were stimulated with 4HT for 3 h and induction of BATF mRNA was analysed by RNA blot hybridization Figure 3 (a) M1 cells were treated with 50 mM PD98059 and stimulated with LIF for 1 h. Cell lysates were immunoblotted with anti-phospho-MAPK antibody (apMAPK) (Cell Signaling) or anti-MAPK antibody (aMAPK) (Santa Cruz Biotechnology). (b) M1 cells were stimulated with LIF plus 50 mM PD98059 or (c) LIF plus 10 ng/ml cyclohexamide (CHX) and induction of BATF mRNA was examined by RNA blot hybridization Oncogene (legend overleaf) Oncogene 1f), there is a transient and reproducible decrease in AP-1 luciferase activity.

Techniques: Northern blot, Hybridization

Journal: Frontiers in Immunology

Article Title: Inhibition of Angiopoietin-2 Production by Myofibrocytes Inhibits Neointimal Hyperplasia After Endoluminal Injury in Mice

doi: 10.3389/fimmu.2018.01517

Figure Lengend Snippet: Impact of coagulation proteases on myofibrocyte phenotype. In (A,B) , responses of wild-type (WT) CD34+ cells are shown as white bars, whereas isolated CD31+ myofibrocytes from CD31-TFPI-Tg mice are shown as black bars. (A) Cells were incubated with FX in presence or absence of FVIIa and FII (prothrombin) plus FVa. Functional tissue factor on WT cells is illustrated by thrombin generation, angiopoietin-2 (Ang-2) secretion, and CXCL-12 secretion. The presence of human tissue factor pathway inhibitor on purified CD31+ myofibrocytes from CD31-TFPI-Tg mice significantly inhibits all three phenotype changes. (B) Proliferation, assessed by 3 H-thymidine incorporation and expressed as counts per minute (CPM) after incubation with FX and FII in presence of FVIIa. (C) Angiopoietin-2 secretion by WT CD34+ cells (3 × 10 4 /well) after 24 h incubation with either PAR-1 antagonist (black bars), PAR-2 antagonist (white bars), or PAR-4 antagonist (gray bars) at the indicated concentrations for 30 min before addition of FVIIa with FX (both 10 nM) with or without prothrombin (4 nM) and FVa (6 nM) as indicated. All conditions performed in triplicate wells. Error bars indicate SEM. In comparison of increasing concentrations of antagonists with FVIIa + FX, p = 0.027 for PAR2, but p = NS for PAR1 and PAR4. In comparison of increasing concentrations of antagonists with FVIIa + FX + FII + FVa, p = 0.05 for PAR1, but p = not significant (NS) for PAR2 and PAR4. Analysis by one-way ANOVA Kruskal–Wallis test. (D) Angiopoietin-2 secretion by WT CD34+ cells (3 × 10 4 /well) after 24 h incubation with PAR-1, -2, or -4 agonists at the indicated concentrations. All conditions performed in triplicate wells. Error bars indicate SEM. p = 0.017 for comparisons of increasing concentrations of PAR1 agonist, p = 0.012 for PAR2, but p = NS for PAR4 agonist. Analysis by one-way ANOVA Kruskal–Wallis test. (E) Dissection of signaling pathways involved in angiopoietin-2 secretion by WT CD34+ cells induced by 24 h incubation with 10 mM PAR-1 or -2 agonists. Cells were incubated with the agonists with or without 50 mM mitogen-activated protein kinase inhibitor PD98059, 10 mM p38-MAPK inhibitor SB203580, 20 mM NF-kB inhibitor SN50, or 1 mM of the S6K1 inhibitor as indicated. All conditions performed in triplicate wells. Error bars indicate SEM. p = 0.05 for PAR-1 agonist without inhibitor vs. +PD98509 and vs. +SB203580. p = > 0.05 all other comparisons. Analysis by Mann–Whitney T test. All experiments repeated at least twice.

Article Snippet: To assess PAR-induced cell signaling, starved cells were treated with 0–20 µM of either PAR-1, PAR-2, or PAR-4 antagonists (Peptides International, Louisville, KY, USA) for 30 min before stimulation with FVIIa + FX or FVIIa + FX + FII at indicated doses; or cells were stimulated with 0–100 µM of PAR-1, PAR-2, or PAR-4 agonist (Peptides International); or cells were treated first with or without 50 µM mitogen-activated protein kinase inhibitor PD98059, 10 µM p38-MAPK inhibitor SB203580, 20 µM NF-kB inhibitor SN50, and 1 µM of the S6K1 inhibitor (All from Merck Millipore, Hertfordshire, UK) for 30 min and then stimulated with 10 µM of PAR-1, PAR-2, or PAR-4 agonist.

Techniques: Coagulation, Isolation, Incubation, Functional Assay, Purification, Dissection, MANN-WHITNEY